US10519249B2 - Covalently linked polypeptide toxin-antibody conjugates - Google Patents

Covalently linked polypeptide toxin-antibody conjugates Download PDF

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US10519249B2
US10519249B2 US15/200,629 US201615200629A US10519249B2 US 10519249 B2 US10519249 B2 US 10519249B2 US 201615200629 A US201615200629 A US 201615200629A US 10519249 B2 US10519249 B2 US 10519249B2
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antibody
hapten
amino acid
polypeptide
seq
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US20170058051A1 (en
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Ulrich Brinkmann
Eike Hoffmann
Stefan Dengl
Klaus Mayer
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Roche Diagnostics GmbH
Hoffmann La Roche Inc
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Hoffmann La Roche Inc
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Definitions

  • complexes comprising a polypeptide toxin and an antibody whereby the components of the complex are covalently linked to each other via a single bond. Also reported are methods for producing the covalent complexes and uses thereof.
  • hapten-based delivery platforms A limitation of currently available hapten-based delivery platforms is the non-covalent linkage between hapten and the delivery vehicle. In applications for which a stable connection between delivery vehicle and payload is desired, non-covalent delivery vehicle-payload complexes may be unsuitable because non-covalently linked payloads may dissociate from the delivery vehicle.
  • One approach is to fuse the payload to entities which stabilized the payload.
  • entities which stabilized the payload.
  • examples of such entities are human serum albumin or human immunoglobulin Fc-regions.
  • This approach is applicable to many linear polypeptides that are composed of naturally occurring amino acid residues and that tolerate modifications at either their C- or N-terminus without losing their biological activity.
  • Polypeptides that are cyclic, stapled, contain non-natural amino acid residues, or additional modifications cannot be recombinantly produced as fusion polypeptides.
  • polypeptides may be the desired choice for therapeutic applications because they are frequently superior to ‘normal’ linear peptides in terms of protease stability, activity and specificity. But these fusions miss a targeted delivery.
  • a major disadvantage of most existing chemical coupling technologies for stabilization or PK modulation of therapeutic polypeptides is their complexity. Beside the chemical coupling step the methods result in many cases in a mixture of polypeptide derivatives that are connected to the PK-modulating entity with uncertain stoichiometries and/or at undefined positions. Additionally currently used polypeptide modification-technologies often result in strongly reduced or even complete loss of biological activity of the therapeutic polypeptide. In addition, it is difficult to predict pharmacological properties and/or possible degradation routes of the chemical conjugates.
  • U.S. Pat. No. 5,804,371 reports hapten-labeled peptides and their use in an immunological method of detection.
  • a digoxigenin-labeled peptide (Bradykinin) and its application to chemiluminoenzyme immunoassay of Bradykinin in inflamed tissues are reported by Decarie A., et al. (Peptides 15 (1994) 511-518).
  • a stabilization and PK-property improvement of the polypeptide toxin can be achieved.
  • This covalent bond is formed between a first cysteine residue introduced into the variable region of the anti-hapten antibody (artificial antibody cysteine residue) and a second cysteine residue present or introduced into the polypeptide toxin ((artificial) polypeptide cysteine residue).
  • the artificial antibody cysteine residue is located in one of the CDRs of the anti-hapten antibody, but does not interfere with the hapten binding properties of the anti-hapten antibody.
  • the (artificial) polypeptide cysteine residue is in close proximity (close spatial distance) to the artificial antibody cysteine residue when the hapten of the haptenylated polypeptide toxin is bound by the anti-hapten antibody. This allows the formation of a covalent bond between the haptenylated polypeptide toxin and the anti-hapten antibody.
  • the (artificial) polypeptide cysteine residue can be included into the amino acid sequence of the polypeptide toxin and, thus, is in the coding region of the polypeptide toxin itself, as opposed to introducing the cysteine residue into the linker that connects the polypeptide toxin and the hapten.
  • both the artificial antibody cysteine residue and the (artificial) polypeptide cysteine residue are at least partially ‘blocked’ in a disulfide with another cysteine residue or glutathione. Nevertheless and very surprisingly it has been found that upon mixture of the recombinantly produced artificial cysteine-containing anti-hapten antibody and the (artificial) cysteine-containing haptenylated polypeptide toxin without additional reagents required a stable disulfide bond is formed by a spontaneous positioned redox-shuffling reaction.
  • covalent antibody-polypeptide toxin conjugates are fully functional in terms of binding and delivery specificities (targeting) as well as polypeptide toxin functionality (i.e. cytotoxic activity towards tumor cells) with the advantage of being more stable than non-covalent complexes in the circulation. It has been found that the disulfide bond linking the antibody and the polypeptide toxin is cleaved inside cells and, thus, the polypeptide toxin is liberated specifically inside cells from the covalent complex.
  • One aspect as reported herein is a (covalent) conjugate of a haptenylated polypeptide and an anti-hapten antibody, wherein a disulfide bond is formed between a cysteine residue either before or after the lysine residue that is used for hapten-conjugation of the polypeptide and a cysteine residue in the CDR2 of the antibody, whereby the CDR2 is determined according to Kabat.
  • polypeptide is a polypeptide toxin.
  • cysteine residue is between 1 to 3 residues before or after the lysine residue that is used for hapten-conjugation. In this embodiment the cysteine residue is at one of the positions N ⁇ 3, N ⁇ 2, N ⁇ 1, N+1, N+2 or N+3 relative to the lysine residue (N).
  • cysteine residue is two residues before (i.e. at position N ⁇ 2 relative to the lysine residue) or after (i.e. at position N+2 relative to the lysine residue) the lysine residue that is used for hapten-conjugation.
  • the lysine residue that is used for hapten-conjugation is within the ten N-terminal amino acid residues of the polypeptide.
  • polypeptide comprises exactly one lysine residue in its amino acid sequence.
  • One aspect as reported herein is a (covalent) conjugate of a haptenylated polypeptide toxin and an anti-hapten antibody, wherein a disulfide bond is formed between a cysteine residue either between 1 to 3 residues before or after the lysine residue that is used for hapten-conjugation of the polypeptide toxin and a cysteine residue in the CDR2 of the antibody, whereby the CDR2 is determined according to Kabat.
  • One aspect as reported herein is a (covalent) conjugate of a haptenylated polypeptide and an anti-hapten antibody, whereby a disulfide bond is formed between a cysteine residue in the polypeptide and a cysteine residue in the CDR2 of the antibody, whereby the CDR2 is determined according to Kabat, characterized in that the polypeptide comprises exactly one lysine residue in its amino acid sequence.
  • polypeptide is a polypeptide toxin.
  • cysteine residue in the polypeptide which is part of the disulfide bond, is either before or after the lysine residue that is used for hapten-conjugation.
  • cysteine residue is between 1 to 3 residues before or after the lysine residue that is used for hapten-conjugation. In this embodiment the cysteine residue is at one of the positions N ⁇ 3, N ⁇ 2, N ⁇ 1, N+1, N+2 or N+3 relative to the lysine residue (N).
  • cysteine residue is two residues before (i.e. at position N ⁇ 2 relative to the lysine residue) or after (i.e. at position N+2 relative to the lysine residue) the lysine residue that is used for hapten-conjugation.
  • the lysine residue that is used for hapten-conjugation is within the ten N-terminal amino acid residues of the polypeptide.
  • Any hapten can be used in the conjugates and methods as reported herein upon derivatization with a linker which allows for the correct spatial orientation of the cysteine residue in the polypeptide ((artificial) polypeptide cysteine residue) and the cysteine residue in the CDR2 of the antibody (artificial antibody cysteine residue) between which the disulfide bond is formed.
  • the anti-hapten antibody specifically binds to the hapten of the haptenylated polypeptide (anti-hapten antibody).
  • the CDR2 is the heavy chain CDR2.
  • the haptenylated polypeptide comprises a hapten, a linker and a polypeptide. In one embodiment the polypeptide is further conjugated to a payload.
  • polypeptide is a polypeptide toxin. In one embodiment the polypeptide toxin is PE25.
  • cysteine residue in the heavy chain CDR2 of the antibody is at position 52, or position 52a, or position 52b, or position 52c, or position 52d, or position 53 according to the heavy chain variable domain numbering of Kabat.
  • cysteine residue in the heavy chain CDR2 of the antibody is at position 52a, or position 52b, or position 52c, or position 53 according to the heavy chain variable domain numbering of Kabat.
  • cysteine residue in the heavy chain CDR2 of the antibody is at position 52b or at position 53 according to the heavy chain variable domain numbering of Kabat.
  • the antibody is a bispecific antibody comprising a first binding specificity to a non-hapten antigen and a second binding specificity to a hapten.
  • the non-hapten antigen is a cell surface antigen. In one embodiment the cell surface antigen is a tumor associated antigen.
  • the bispecific antibody is a full length antibody.
  • one heavy chain of the bispecific antibody comprises a hole mutation and the respective other chain comprises a knob mutation.
  • the bispecific antibody is a full length antibody to which at each C-terminus a scFv or a scFab is fused either directly or via a peptidic linker.
  • the antibody is a humanized or a human antibody.
  • the constant region of the antibody is of the IgG1 subclass or of the IgG4 subclass.
  • the antibody has a constant region of the IgG1 subclass with an alanine at position 234 and 235 and with a glycine at position 329 with numbering according to the EU index of Kabat.
  • the antibody has a constant region of the IgG4 class with a proline at position 228, a glutamic acid at position 235 and a glycine at position 329 with numbering according to the EU index of Kabat.
  • the conjugate comprises exactly one disulfide bond per heavy chain CDR2.
  • the disulfide bond is formed without the addition of redox active agents.
  • the antigen or the hapten is conjugated to the polypeptide via a linker.
  • the linker is a non-peptidic linker.
  • the linker is a carboxymethyl-linker or a caproic acid linker.
  • the hapten is biotin, or theophylline, or digoxigenin, or carborane, or fluorescein, or bromodeoxyuridine. In one embodiment the hapten is biotin or digoxigenin.
  • One aspect as reported herein is a pharmaceutical formulation comprising the conjugate as reported herein and a pharmaceutically acceptable carrier.
  • One aspect as reported herein is the use of a conjugate as reported herein in the manufacture of a medicament.
  • One aspect as reported herein is the use of a conjugate as reported herein to increase the stability of the polypeptide.
  • One aspect as reported herein is the use of a conjugate as reported herein to reduce or eliminate off-target toxic effects of the polypeptide.
  • One aspect as reported herein is the use of a conjugate as reported herein to increase the activity of the polypeptide.
  • One aspect as reported herein is the use of a conjugate as reported herein to increase the in vivo half-life of the polypeptide.
  • One aspect as reported herein is the use of a conjugate as reported herein in the treatment of a disease.
  • One aspect as reported herein is a method of treating an individual having a disease comprising administering to the individual an effective amount of a conjugate as reported herein.
  • One aspect as reported herein is a method of treating a disease in an individual comprising administering to the individual an effective amount of the conjugate as reported herein.
  • the disease is cancer.
  • One aspect as reported herein is a method of producing a conjugate as reported herein comprising the combination of an antibody comprising an artificial antibody cysteine residue and a haptenylated polypeptide comprising an (artificial) polypeptide cysteine residue, whereby the alpha carbon atom of the artificial antibody cysteine residue is about 10 to 11 Angstrom apart from the atom of the polypeptide toxin to which the linker is fused.
  • One aspect as reported herein is a method of producing a conjugate as reported herein comprising the steps of
  • bispecific antibody for targeted delivery of a haptenylated compound to a target cell, wherein the bispecific antibody comprises a first binding site that specifically binds to the haptenylated polypeptide and a second binding specificity that specifically binds to a cell surface marker of the cell.
  • a disulfide bond is formed between a cysteine residue either before or after the lysine residue that is used for hapten-conjugation of the polypeptide and a cysteine residue in the CDR2 of the antibody, whereby the CDR2 is determined according to Kabat.
  • cysteine residue is between 1 to 3 residues before or after the lysine residue that is used for hapten-conjugation. In this embodiment the cysteine residue is at one of the positions N ⁇ 3, N ⁇ 2, N ⁇ 1, N+1, N+2 or N+3 relative to the lysine residue.
  • cysteine residue is two residues before (i.e. at position N ⁇ 2 relative to the lysine residue) or after (i.e. at position N+2 relative to the lysine residue) the lysine residue that is used for hapten-conjugation.
  • the lysine residue that is used for hapten-conjugation is within the ten N-terminal amino acid residues of the polypeptide.
  • polypeptide comprises exactly one lysine residue in its amino acid sequence.
  • FIGS. 1A-1C Comparison of the binding of recombinant humanized anti-biotin antibodies with and without introduced VH53C mutation. Binding properties were analyzed by surface plasmon resonance (SPR) technology using a BIAcore T100 or BIAcore 3000 instrument.
  • FIG. 2 Introduction of SH functionalities in the hapten as well as in the antibody at appropriate positions allow the antibody and the hapten to form a covalent bond in between resulting in a conjugate.
  • FIGS. 3A-3B Scheme of SDS-PAGE self-fluorescence band pattern (without further staining of the SDS-PAGE gel):
  • FIG. 3A If no covalent bond is formed between the antibody and the hapten-fluorophore conjugate both under reducing or non-reducing conditions one self-fluorescent band at the molecular weight of free hapten-fluorophore conjugate can be detected.
  • FIG. 3B If a covalent bond is formed between the antibody and the hapten-fluorophore conjugate under non-reducing conditions one self-fluorescent band at the combined molecular weight of the antibody and the hapten-fluorophore conjugate can be detected. Under reducing conditions the disulfide bridges in the conjugate of the antibody and the hapten-fluorophore conjugate (haptenylated compound) are cleaved and one self-fluorescent band at the molecular weight of free hapten-fluorophore conjugate can be detected.
  • FIG. 4 Conjugate formation of hapten-binding Cys-mutated antibodies with hapten-Cys-fluorescent label conjugates (haptenylated compound) in the presence of redox active agents: oxidation agent (glutathione disulfide, GSSG) and reducing agent (dithioerythritol, DTE): Antibody complexation and subsequent covalent linkage at defined positions is detected by fluorescence signals in SDS PAGE analyses. Non-reducing (upper images) and reducing (lower images) SDS-PAGE analyses were performed as described in Example 6. Covalently antibody linked haptens are detectable as larger sized protein bound signals at the appropriate positions under non-reduced conditions. These signals detach from protein upon reduction and are visible as small entities under reducing conditions.
  • FIG. 5 Complex formation of hapten-binding Cys mutated antibodies with hapten-Cys-fluorescent label conjugates in the presence solely of an oxidation agent (glutathione disulfide, GSSG) but in the absence of reducing agents or in the absence of both: Antibody complexation and subsequent covalent linkage at defined positions is detected by fluorescence signals in SDS PAGE analyses. Non-reducing (upper images) and reducing (lower images) SDS-PAGE analyses were performed as described in Example 7. Covalently antibody linked haptens are detectable as larger sized protein bound signals at the appropriate positions under non-reduced conditions. These signals detach from protein upon reduction and are visible as small entities under reducing conditions.
  • an oxidation agent glutasulfide
  • FIG. 6 X-ray structure of murine anti-biotin antibody in complex with biocytinamid. Amino acid residues that are interacting with biocytinamid are shown in a stick representation.
  • FIGS. 7A-7B Results of in vivo blood PK study with covalent conjugates and non-covalent complexes compared to non-complexed antigen/hapten; the relative remaining fluorescence intensity (%, solid marks) of Cy5-mediated fluorescence of Biotin-Cy5 non-covalent complexes ( FIG. 7A ) and covalent (SS-bridged) conjugates ( FIG.
  • FIG. 8 Western blot of the determination of the amount of digoxigenylated PYY polypeptide in the serum of mice.
  • FIG. 9 Analysis of affinity-driven complexation of haptenylated compounds with anti-hapten antibodies.
  • Antibody complexation and subsequent covalent linkage at defined positions is directed by fluorescence signals in SDS PAGE analyses, which were carried out as described in Example 14.
  • the white arrows mark the excess (uncoupled) biotin-Cys-Cy5, which is significantly higher when anti-digoxigenin antibody VH52bC is used, because the conjugation reaction is not affinity driven in this case.
  • FIGS. 10A-10E Cysteine positions and disulfide patterns within the Fab region, required to form a Dig-binding antibody with additional cysteine at position 52b for hapten-mediated site-directed directed covalent payload coupling.
  • FIG. 10A Cysteines and disulfide pattern in VH and CH1 domains, and in VL and CL domains that are required to form functional Fab fragments.
  • FIG. 10B Cysteines and disulfide pattern in VH and CH1 domains, and in VL and CL domains that are required to form functional Fab fragments with additional cysteine at position 52b for hapten-mediated site-directed directed covalent payload coupling.
  • FIGS. 10A-10E Cysteine positions and disulfide patterns within the Fab region, required to form a Dig-binding antibody with additional cysteine at position 52b for hapten-mediated site-directed directed covalent payload coupling.
  • FIG. 10A Cysteines and disulfide pattern in VH and CH
  • FIG. 10C-10D Potential to form incorrect disulfide bonds within the VH domain of the VH52b variant which would result in misfolded nonfunctional antibodies.
  • FIG. 10E Example for a potential incorrect interdomain disulfide bond within the Fv region of the VH52b variant, which would result in misfolded nonfunctional antibodies.
  • FIGS. 11A-11E Cysteine positions and disulfide patterns required to form a Dig-binding disulfide-stabilized single-chain Fv with additional cysteine at position 52b for hapten-mediated site-directed directed covalent payload coupling.
  • FIG. 11A Cysteines in VH and VL domains that are required to form functional scFvs, dsscFvs and 52b mutated dsscFvs.
  • FIG. 11B correct pattern of disulfide bonds that must be formed to generate functional scFvs, dsscFvs and 52b mutated dsscFvs.
  • FIG. 11A Cysteines in VH and VL domains that are required to form functional scFvs, dsscFvs and 52b mutated dsscFvs.
  • FIG. 11B correct pattern of disulfide bonds that must be formed to generate functional scFvs, ds
  • FIG. 11C Potential to form incorrect disulfide bonds which would result in misfolded nonfunctional scFvs.
  • FIG. 11D Potential to form incorrect disulfide bonds which would result in misfolded nonfunctional dsscFvs.
  • FIG. 11E Potential to form incorrect disulfide bonds which would result in misfolded nonfunctional 52b mutated dsscFvs.
  • FIG. 12 Composition of a LeY-Dig bispecific antibody derivative as delivery vehicle for covalently coupled payloads.
  • FIGS. 13A-13C Expression and Purification of bispecific anti-hapten antibody derivatives for targeted delivery of covalently coupled payloads.
  • FIG. 13A For Western blot analyses, cell culture supernatants were subjected to SDS PAGE (NuPAGE® 4-12% Bis-Tris Gel (1.0 mm ⁇ 12 well) (Invitrogen; Cat. No. NP0322) and proteins were subsequently transferred to IMMOBILON® Transfer Membranes (IMMOBILON®), PVDF with pore Size: 0.45 ⁇ m. Antibody derivatives were detected by Anti-Human Kappa Light Chain)-Alkaline Phosphatase antibody produced in goat, (affinity purified), Sigma (Cat. No.
  • FIG. 13B SDS-PAGE analyses (NuPAGE® 4-12% Bis-Tris Gel and subsequent staining with Coomassie brilliant blue demonstrates purity of protein preparations and visualizes polypeptide chains related to the IgG with the apparent molecular sizes that correspond to their calculated molecular weights.
  • Lane 1 molecular weight marker
  • Lane 2 LeY-Dig(52bC) bispecific antibody with extended H-chain reduced
  • lane 3 LeY-Dig(52bC) bispecific antibody with extended heavy-chain non-reduced
  • Lane 1 molecular weight marker
  • Lane 2 LeY-Dig(52bC) bispecific antibody with extended H-chain reduced
  • lane 3 LeY-Dig(52bC) bispecific antibody with extended heavy-chain non-reduced
  • FIG. 13C Size exclusion chromatography (Superdex 200) demonstrates homogeneity and lack of aggregates in the protein preparations of the LeY-Dig(52bC) bispecific antibody derivative after Protein A purification.
  • FIG. 15 Pharmacokinetics under in vivo-like conditions of Cy5-mediated fluorescence of Biotin-Cy5 of non-covalent complexes and of covalent (disulfide-bridged) conjugates, as well as of non-complexed Biotin-Cy5, determined by non-invasive eye imaging; solid diamond: biotin-Cy5, solid square biotin-Cy5+anti-biotin antibody (complex); triangle: Cy5-Biotin-anti-biotin antibody conjugate.
  • FIGS. 16A-16C FIG. 16A : Composition, structure and molecular weight of Theophylline-Cys-Cy5;
  • FIG. 16B Size exclusion chromatography demonstrates purity and homogeneity of purified theophylline-binding antibody variants; peak #2 shows the purified product, lack of peak #1 indicates that such preparations are free of aggregates;
  • FIG. 16A Composition, structure and molecular weight of Theophylline-Cys-Cy5
  • FIG. 16B Size exclusion chromatography demonstrates purity and homogeneity of purified theophylline-binding antibody variants
  • peak #2 shows the purified product, lack of peak #1 indicates that such preparations are free of aggregates;
  • FIG. 16A Composition, structure and molecular weight of Theophylline-Cys-Cy5
  • FIG. 16B Size exclusion chromatography demonstrates purity and homogeneity of purified theophylline-binding antibody variants
  • peak #2 shows the purified
  • 16C formation of covalent complexes between theophylline-binding antibodies and Theophylline-Cys-Cy5 as demonstrated by non-reducing (left lanes) and reducing (right lanes) SDS PAGE; Cy5 appears coupled to the H-chain under non-reducing conditions only in samples that contained Theophylline-Cys-Cy5 and Cys-mutated antibody, these covalent conjugates disintegrate upon reduction (right lanes); Lanes 1: Molecular weight marker; 2-4 non-reducing—2: anti-Theophylline antibody (without Cys-mutation)+Theophylline-Cys-Cy5 (complex); 3: anti-Theophylline antibody-cys_55+Theophylline-Cys-Cy5 (conjugate); 4: anti-Theophylline antibody-cys_54+Theophylline-Cys-Cy5 (conjugate); 5-7 reducing 5: anti-Theophylline antibody (without Cys-mut
  • FIG. 17 Formation of covalent complexes between biotin-binding antibodies and Biotin-Cys-Cy5 is demonstrated by non-reducing and reducing SDS PAGE; the coupling reaction was performed in murine serum at 37° C. for 1 hr.
  • Cy5 appears coupled to the H-chain under non-reducing conditions only in samples that contained Biotin-Cys-Cy5 and Cys-mutated antibody; these covalent conjugates disintegrate upon reduction (right lanes); lanes 1: Molecular weight marker; 2-3 non-reducing—2: anti-Biotin antibody (without Cys mutation)+Biotin-Cys-Cy5 (complex); 3: anti-Biotin antibody-Cys+Biotin-Cys-Cy5 (conjugate); 4-5 reducing—5: anti-Biotin antibody (without Cys mutation)+Biotin-Cys-Cy5 (complex); 6: anti-Biotin antibody-Cys+Biotin-Cys-Cy5 (conjugate).
  • FIG. 18 In vivo pharmacokinetics of Cy5-mediated fluorescence of Biotin-Cy5 of non-covalent complexes and of covalent (disulfide-bridged) conjugates, as well as of non-complexed Biotin-Cy5, determined by non-invasive eye imaging; solid diamond: biotin-Cy5, solid circle: biotin-Cy5 administered 24 hours after administration of anti-biotin antibody (in vivo complex formation); solid square: biotin-Cys-Cy5 administered 24 hours after administration of anti-biotin antibody-Cys (in vivo conjugate formation).
  • FIG. 19 The protein structure of murine anti-Biotin antibody-Fab-fragment was determined in complex with biocytinamid: the complexed hapten is positioned in close proximity to a negatively charged cluster of amino acids; biotin which—as hapten—is derivatized for payload coupling at its carboxyl group binds with good efficacy as there is no charge repulsion at this position (due to the lack of the COOH group); in contrast, free (normal) biotin cannot bind efficient to the antibody because its carboxyl group would be in close proximity to this negative charge cluster, and hence becomes repulsed.
  • FIGS. 20A-20B FIG. 20A : Scheme of anti-hapten bispecific antibodies for targeted delivery of polypeptide toxins; disulfide-stabilized scFvs that bind haptens are recombinantly fused to the heavy chains (C-terminus of the CH3 domain), alternatively it is also possible to fuse to C-termini of Fab fragments or to other positions of recombinant binding modules.
  • Bispecific antibody encoding sequences were generated by gene synthesis (Geneart, Germany), subcloned into expression vectors and produced and purified as described (Metz, S., et al., Proc. Natl. Acad. Sci. USA 108 (2011) 8194-8199).
  • a humanized dsscFv of the 19-11 antibody (Metz et al. and 27209 WO) was used as digoxigenin-binding entity; VH and VL were introduced into the Fab arms of the IgG and Fab-formats sequences were derived from the LeY-binding antibody B3 (see Metz et al.).
  • FIG. 20B Expression and Purification of bispecific antibodies: SEC profiles and SDS-PAGE demonstrating the purity and homogeneity of bispecific antibody preparations; for transient expression, plasmids encoding light and heavy chains or of the Fab-Fv fusions were co-transfected into HEK293 cells were cultivated in serum free medium, supernatants were clarified seven days after transfection by centrifugation and 0.22 ⁇ m filtration, bispecific antibodies were purified by protein A (IgG-Fv) followed by SEC (Superdex200 HiLoad 26/60, GE Healthcare) equilibrated with 20 mM histidine, 140 mM NaCl, at pH 6.0, protein concentrations were determined by optical density at 280 nm with 320 nm as background, homogeneity of purified proteins was confirmed by SDS-PAGE.
  • protein A IgG-Fv
  • SEC Superdex200 HiLoad 26/60, GE Healthcare
  • FIGS. 21A-21C FIG. 21A : Derivatives of Pseudomonas exotoxin (PE); domain composition of PE and PE variants, and generation of the novel variant PE25 (NLys-PE25SQ ⁇ ) for site-directed digoxigenylation; PE25 becomes digoxigenylated with Dig-NHS at primary amino groups of lysine side chains; the N-terminus of the proteins can also be a target for NHS-mediated digoxigenin-conjugation; PE25 (NLysPE25SQ ⁇ ) contains the amino acid sequence (N-term) NH2-MLQGTKLMAEE (SEQ ID NO: 193) fused to the amino acids 274-284 (domain II processing site), followed by amino acids 394-612 (domain III) of PE (pdb 1IKQ_A); in addition, positions 406, 432, 467, 490, 513, 548, 590, 592 were mutated as in the LR8M toxin derivative (Hansen, J
  • the lysine at position 606 was mutated to glutamine and the last amino acid of PE (Lysine 613) was deleted; the amino-terminal sequence was altered by exchanging the Lys residue to Ser in S-PE25, by exchanging the Gly residue to Cys in NCK-PE25, and by exchanging the Met residue to Cys in NKC-PE25; the coding sequences for these derivatives were generated by gene synthesis (Geneart, Germany) or mutagenesis, and inserted into vectors for inducible expression in E. coli.
  • FIG. 21B Expression and Purification of Pseudomonas exotoxin derivatives: The proteins became secreted into the periplasm and were purified applying techniques that were previously described (Debinski, W. and Pastan, I., Cancer Res. 52 (1992) 5379-5385; Debinski, W. and Pastan, I., Bioconjug. Chem.
  • periplasm preparations generated via osmotic shock from harvested bacteria were loaded to Q-SEPHAROSE® to capture the polypeptide toxin; polypeptide toxin was eluted with a salt gradient, subjected to SEC to obtain monomeric polypeptide toxin with low levels of aggregates and smaller protein contaminants; SEC was performed and toxin fractions were stored in PBS. Homogeneity of purified proteins was confirmed by SEC and SDS-PAGE.
  • FIG. 21C N-terminal sequences of PE25, S-PE25, NKC-PE25 and NCK PE25: the N-terminal methionine is encoded by the ATG start codon is indicated in brackets because it may become removed by posttranslational processing in E. coli .
  • the Lysine residue that becomes coupled to the hapten is in highlighted (bold) and the additional Cysteine before or after this Lysine is bold underlined.
  • FIGS. 22A-22C FIG. 22A : Complexation and covalent linkage of antibody and toxin: Reduced and non-reduced samples of covalent complexes comprising NKC-PE25 were separated via SDS-PAGE (NuPAGE® 4-12% Bis-Tris Gel (1.0 mm ⁇ 10 well) and either visualized by Coomassie blue staining, or subjected to Western blot to IMMOBILON® PVDF Transfer Membranes (IMMOBILON®-P), Pore Size: 0.45 ⁇ m (middle and right panel). Western blot analysis with polyclonal anti-PE antibodies (anti- Pseudomonas exotoxin A, antibody produced in rabbit (Sigma, Cat. No.
  • SDS-PAGE NuPAGE® 4-12% Bis-Tris Gel (1.0 mm ⁇ 10 well) and either visualized by Coomassie blue staining, or subjected to Western blot to IMMOBILON® PVDF Transfer Membranes (IMMOBILON®-
  • FIG. 22B Complexation and covalent linkage of anti-digoxigenin antibody and digoxigenylated polypeptide toxin: a model of the components of digoxigenylated PE25 (Dig-PE25) complexed with the a digoxigenin-binding Fv was generated by linking the structural model of the digoxigenin-spacer::anti-digoxigenin antibody structure (PDB_3RA7, 1) with that of PE25 (PDB_1K2N and 1IKQ, the second part of the linker is taken from likq); an appropriately sized digoxigenin attached via an amino caproic acid spacer to the singular lysine of PE25 connects both structures; also shown are structure models that show possible configurations of the covalent linkage between the extra cysteines VH of anti-digoxigenin antibody and the cysteine in NKC-PE25 or NCK PE25.
  • FIG. 23 Bispecific antibody-mediated targeted toxin delivery: Cell proliferation (BrdU) assays were performed to analyze toxic effects towards tumor cells; LeY expressing MCF-7 cells were exposed for 48 hrs. to toxin alone (upper panel) or vehicle-toxin complexes in the IgG-Fv Format (lower panel).
  • FIG. 24 Cell targeting bispecific antibodies release hapten-positioned disulfide connected payloads within target cells. Confocal microscopy reveals bsAb-targeted delivery of the disulfide-conjugated Biotin-Cys-Cy5-payload to and into MCF-7 cells. The bsAb is detected by Alexa labeled huFc-binding antibodies, the Bio-Cys-Cy5 payload by its fluorescence. Co-localization of bsAb and payload is indicated by mixed color (3), isolated bsAb appears in color 1 (1) and biotin without antibody in color 2 (2). The images (six hours after LeY-bsAb application to LeY-expressing MCF7 cells) reveal separation of biotin from the complexes at time points where the bsAb vehicle is still sufficiently intact for detection by secondary antibodies.
  • acceptor human framework for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • amino acid denotes the group of carboxy ⁇ -amino acids, either occurring naturally, i.e. which directly or in form of a precursor can be encoded by a nucleic acid, or occurring non-naturally.
  • the individual naturally occurring amino acids are encoded by nucleic acids consisting of three nucleotides, so called codons or base-triplets. Each amino acid is encoded by at least one codon. This is known as “degeneration of the genetic code”.
  • amino acid denotes the naturally occurring carboxy ⁇ -amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophane (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).
  • non-naturally occurring amino acids include, but are not limited to, Aad (alpha-Aminoadipic acid), Abu (Aminobutyric acid), Ach (alpha-aminocyclohexane-carboxylic acid), Acp (alpha-aminocyclopentane-carboxylic acid), Acpc (1-Aminocyclopropane-1-carboxylic acid), Aib (alpha-aminoisobutyric acid), Aic (2-Aminoindane-2-carboxylic acid; also called 2-2-Aic), 1-1-Aic (1-aminoindane-1-carboxylic acid), (2-aminoindane-2-carboxylic acid), Allylglycine (AllylGly), Alloisoleucine (allo-Ile), Asu (alpha-Aminosuberic acid, 2-Aminooctanedioc acid), Bip (4-phenyl-phenylalanine-carbox
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • antibody fragment denotes a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′) 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • biotin short “BI”, denotes 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d[imidazol-4-yl]pentanoic acid. Biotin is also known as vitamin H or coenzyme R.
  • bispecific antibodies denotes antibodies which have two different (antigen/hapten) binding specificities.
  • bispecific antibodies as reported herein are specific for two different antigens, i.e. a hapten and a non-hapten antigen.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • polypeptide toxin refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Polypeptide toxins include, but are not limited to, enzymes and fragments thereof such as nucleolytic enzymes; toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, cytotoxins (e.g. Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and the like), enzymes, growth factors, transcription factors.
  • digoxigenin short “DIG”, denotes 3-R3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydro-cyclopenta[a]-phenanthren-17-yl]-2H-furan-5-one (CAS number 1672-46-4).
  • Digoxigenin is a steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata (foxgloves) (Polya, G., Biochemical targets of plant bioactive compounds, CRC Press, New York (2003) p. 847).
  • effector functions denotes those biological activities attributable to the Fc-region of an antibody, which vary with the antibody class.
  • antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • an agent e.g., a pharmaceutical formulation
  • an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result denotes an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′) 2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
  • F(ab′) 2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Fc-region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc-regions and variant Fc-regions.
  • a human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), NIH Publication 91-3242.
  • fluorescein short “FLUO”, denotes 6-hydroxy-9-(2-carboxyphenyl)-(3H)-xanthen-3-on, alternatively 2-(6-hydroxy-3-oxo-(3H)-xanthen-9-yl)-benzoic acid.
  • Fluorescein is also known as resorcinolphthalein, C.I. 45350, solvent yellow 94, D & C yellow no. 7, angiofluor, Japan yellow 201, or soap yellow.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • artificial cysteine residue denotes a cysteine amino acid residue which has been engineered into a parent antibody or polypeptide toxin, which has a thiol functional group (SH), and is not paired in an intramolecular disulfide bridge. Nevertheless, a free cysteine amino acid can be paired as intermolecular disulfide bridge, e.g. with glutathione.
  • full length antibody denotes an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc-region as defined herein.
  • Native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain.
  • VH variable region
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • a “full length antibody” is an antibody comprising a VL and VH domain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or an amino acid sequence variant thereof.
  • the full length antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc-region or amino acid sequence variant Fc-region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B-cell receptor and BCR.
  • hapten denotes a small molecule that can elicit an immune response only when attached to a large carrier such as a protein.
  • exemplary haptens are aniline, o-, m-, and p-aminobenzoic acid, quinone, histamine-succinyl-glycine (HSG), hydralazine, halothane, indium-DTPA, fluorescein, biotin, digoxigenin, theophylline and dinitrophenol, and bromodeoxyuridine.
  • HSG histamine-succinyl-glycine
  • halothane halothane
  • indium-DTPA fluorescein
  • biotin digoxigenin
  • theophylline theophylline and dinitrophenol
  • bromodeoxyuridine bromodeoxyuridine.
  • the hapten is biotin or digoxigenin or theophylline or carborane or bromodeoxyuridine.
  • haptenylated polypeptide toxin denotes a molecule consisting of a hapten which is covalently linked to a polypeptide toxin. Activated hapten derivatives are often used as starting materials for the formation of such conjugates.
  • the hapten is digoxigenin and it is conjugated (in one embodiment via its 3-hydroxy group) to the polypeptide toxin via a linker.
  • the linker comprises a) one or more (in one embodiment three to six) methylene-carboxy-methyl groups (—CH 2 —C(O)—), and/or b) from 1 to 10 (in one embodiment from 1 to 5) amino acid residues (in one embodiment selected from glycine, serine, glutamate, ⁇ -alanine, ⁇ -aminobutyric acid, ⁇ -aminocaproic acid or lysine), and/or c) one or more (in one embodiment one or two) compounds having the structural formula NH 2 —[(CH 2 ) n O] x CH 2 —CH 2 —COOH in which n is 2 or 3 and x is 1 to 10, in one embodiment 1 to 7.
  • the last element results (at least partly) in a linker (part) of the formula —NH—[(CH 2 ) n O] x CH 2 —CH 2 —C(O)—.
  • a linker (part) of the formula —NH—[(CH 2 ) n O] x CH 2 —CH 2 —C(O)—.
  • One example of such a compound is e.g. 12-amino-4,7,10-trioxadodecanoic acid (results in a TEG (triethylenglycol) linker).
  • the linker has a stabilizing and solubilizing effect since it contains charges or/and can form hydrogen bridges. In addition it can sterically facilitate the binding of the anti-hapten antibody to the hapten-conjugated polypeptide toxin.
  • the linker is located at a side chain of an amino acid of the polypeptide toxin (e.g. conjugated to a lysine side chain via an amino group). In one embodiment the linker is located at the amino terminus or at the carboxy terminus of the polypeptide toxin.
  • the position of the linker on the polypeptide is typically chosen at a region where the biological activity of the polypeptide toxin is not affected. Therefore the exact attachment position of the linker depends on the polypeptide toxin and the relevant structure elements which are responsible for the biological activity. The biological activity of the polypeptide toxin to which the hapten is attached can be tested in an in vitro assay.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”), and/or contain the antigen-contacting residues (“antigen contacts”).
  • CDRs complementarity determining regions
  • hypervariable loops form structurally defined loops
  • antigen contacts antigen contacts
  • antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
  • HVRs herein include
  • mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g. cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • an “isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • the term “monospecific antibody” denotes an antibody that has one or more binding sites each of which has the same binding specificity, i.e. binds to the same antigen or hapten.
  • naked antibody refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel.
  • the naked antibody may be present in a pharmaceutical formulation.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • a “parent antibody” is an antibody comprising an amino acid sequence from which one or more amino acid residues are replaced by one or more cysteine residues.
  • the parent antibody may comprise a native or wild-type sequence.
  • the parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions) relative to other native, wild-type, or modified forms of an antibody.
  • the parent antibody binds specifically to a hapten.
  • a parent antibody may be directed additionally also against a target antigen of interest, e.g. a biologically important polypeptide. Antibodies directed against non-polypeptide antigens are also contemplated.
  • fMLP denotes the tripeptide consisting of N-formylmethionine, leucine and phenylalanine.
  • the effector moiety is fMLP or a derivative thereof.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • polypeptide is a polymer consisting of amino acids joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 20 amino acid residues may be referred to as “peptides”, whereas molecules consisting of two or more polypeptides or comprising one polypeptide of more than 100 amino acid residues may be referred to as “proteins”. A polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions, or carboxylic acid esters. The non-amino acid components may be added by the cell, in which the polypeptide is expressed, and may vary with the type of cell. Polypeptides are defined herein in terms of their amino acid backbone structure or the nucleic acid encoding the same.
  • a polypeptide as used in the current invention comprises at least three amino acid residues.
  • One amino acid residue is cysteine residue for the formation of the disulfide bond to the anti-hapten antibody and one amino acid residue is a lysine residue for conjugation to the hapten.
  • the third amino acid residue of the polypeptide in the haptenylated polypeptide is either i) a single amino acid residue for conjugation to the payload or ii) a polypeptide. It is also encompassed that the polypeptide itself is part of a larger polypeptide with biological activity, such as e.g. of a polypeptide toxin.
  • N-terminus refers to the free alpha-amino group of an amino acid in a polypeptide
  • C-terminus refers to the free ⁇ -carboxylic acid terminus of an amino acid in a polypeptide.
  • a polypeptide which is N-terminated with a group refers to a polypeptide bearing a group on the alpha-amino nitrogen of the N-terminal amino acid residue.
  • An amino acid which is N-terminated with a group refers to an amino acid bearing a group on the alpha-amino nitrogen.
  • single-chain Fv short “scFv”, denotes an antibody fragment that comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • theophylline short “THEO”, denotes 1,3-dimethyl-7H-purine-2,6-dione.
  • Theophylline is also known as dimethylxanthine.
  • treatment denotes a clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • x-valent e.g. “mono-valent” or “bi-valent” or “tri-valent” or “tetra-valent”, denotes the presence of a specified number of binding sites, i.e. “x”, in an antibody molecule.
  • the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding site, four binding sites, and six binding sites, respectively, in an antibody molecule.
  • the bispecific antibodies as reported herein are at least “bivalent” and may be “trivalent” or “multivalent” (e.g. “tetravalent” or “hexavalent”).
  • the bispecific antibody as reported herein is bivalent, trivalent, or tetravalent.
  • the bispecific antibody is bivalent.
  • the bispecific antibody is trivalent.
  • the bispecific antibody is tetravalent.
  • the antibodies as reported herein have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent).
  • the term bispecific antibodies includes, for example, multivalent single chain antibodies, diabodies and triabodies, as well as antibodies having the constant domain structure of full length antibodies to which further antigen-binding sites (e.g., single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2) are linked via one or more peptide-linkers.
  • the antibodies can be full length from a single species, or be chimerized or humanized.
  • binding sites may be identical, so long as the protein has binding sites for two different antigens. That is, whereas a first binding site is specific for a hapten, a second binding site is specific for a non-hapten antigen, and vice versa.
  • variable region denotes the domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).
  • vector denotes a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
  • the Pseudomonas exotoxin A chain is a 66 kDa polypeptide consisting of 613 amino acid residues. It is built up of three functional domains: domain I, the N-terminal receptor-binding domain, which binds to eukaryotic cells, domain II, which is responsible for internalization and which becomes proteolytically processed II, and domain III, which is C-terminal and released into the cytoplasm after processing. Domain III ADP-ribosylates eEF2, which causes inhibition of protein synthesis and subsequent cell death.
  • FIG. 21A The composition of different truncated variants of PE are shown in FIG. 21A .
  • NLysPE38 the cell binding domain I and domain IB are deleted. This molecule by itself has a very low cytotoxic potency.
  • NLysPE38 contains a lysine residue close to its N-terminus (Nlys), which can be chemically modified by NHS-chemistry.
  • Nlys N-terminus
  • dsscFv-fusions disulfide stabilized Fv-fusions
  • most of domain II can also be deleted without loss of potency as long as the processing site is retained.
  • the size of the truncated toxin variant within such fusion proteins was approximately 25 kDa.
  • Truncated toxin variants still contain lysine residues in domain III.
  • NlysPE40QQR the lysines of domain III have been replaced by glutamine and/or arginine in order to reduce the risk of inactivation of domain III by amine-modifying reagents such as NHS when e.g. chemically conjugating the truncated toxin variant.
  • amine-modifying reagents such as NHS when e.g. chemically conjugating the truncated toxin variant.
  • NLysPE25SQ ⁇ in which domains I and IB as well as most of domain II have been deleted (toxin moiety of CD22-LR8M), and which contains lysine to serine or glutamine exchanges in domain III.
  • NLysPE25SQ ⁇ is a rather small toxin moiety that contains only one lysine at its N-terminus.
  • the primary amine of this lysine (and that of the N-terminus) can be modified by NHS-reagents without affecting other positions of the toxin.
  • toxin derivatives that contain an extra cysteine for hapten mediated covalent coupling to a bispecific antibody
  • a cysteine was placed into NLysPE25SQ ⁇ either before or after the lysine residue that is used for hapten-conjugation.
  • the invention is based on the finding that a covalent conjugate comprising a haptenylated polypeptide and an anti-hapten antibody that specifically binds to the hapten can be obtained by the formation of a disulfide bond between a properly placed cysteine residue in the polypeptide and a cysteine residue in the variable domain of the antibody, especially in the CDR2 of the antibody, whereby the CDR2 is determined according to heavy chain variable domain numbering according to Kabat.
  • the antigen is a hapten.
  • the hapten is biotin, or digoxigenin, or fluorescein, or theophylline, or carborane.
  • the haptenylated polypeptide is a conjugate comprising a hapten, a linker and a polypeptide toxin.
  • the hapten is biotin, or digoxigenin, or fluorescein, or theophylline, or carborane, or bromodeoxyuridine.
  • the polypeptide is a polypeptide toxin.
  • Covalent conjugates of a haptenylated polypeptide and an anti-hapten antibody may confer benign biophysical behavior and improved PK properties to the polypeptide.
  • the conjugates can be used to target the polypeptide to cells which display the antigen that is recognized by the second binding specificity of the bispecific antibody.
  • Such conjugates are composed of one anti-hapten binding specificity and one (non-hapten) antigen binding specificity.
  • the stoichiometric ratio of antibody molecules to haptenylated polypeptide molecules depends on the format of the bispecific antibody and can be 1:1, 1:2, 2:1, 2:2, 2:4 and 4:2 (antibody:haptenylated polypeptide).
  • the polypeptide retains good biological activity despite being conjugated the hapten, as well as being conjugated to the anti-hapten antibody. It is also desired (in case of bispecific targeting modules) that the cell surface target binding site of the bispecific antibody retains its binding specificity and affinity in the presence of the covalently conjugated haptenylated polypeptide.
  • both compounds have to be modified by the introduction of a reactive group.
  • the two reactive groups are brought in close proximity allowing the formation of a covalent bond.
  • the modification is the introduction of a thiol functionality in each of the compounds.
  • the thiol compound is a cysteine residue.
  • the position to be mutated must concomitantly fulfill two requirements: (i) the coupling positions should be in proximity to the binding region to utilize the hapten positioning effect for directed coupling, and (ii) the mutation and coupling position must be positioned in a manner that hapten binding by itself is not affected.
  • These requirements for finding a suitable position are de facto ‘contradicting’ each other because requirement (i) is best served by a position close to the binding site, while requirement (ii) is most safely achieved by positions that are distant from the binding site.
  • the first position is located at position VH52b or at position VH53, respectively, according to the Kabat numbering of the heavy chain variable domain. If the antibody has a short VH CDR2, which does not have intermittent residues, such as 52a, 52c, 52c, and 52d, the position is 53 (numbering and alignment according to the numbering scheme and rules of Kabat for the antibody heavy chain variable domain). If the antibody has a long VH CDR2 comprising residues 52a and 52b, and optionally further residues as 52c and 52d, etc. the position is 52b (numbering and alignment according to the numbering scheme and rules of Kabat for the antibody heavy chain variable domain).
  • the second position is located at position VH28 according to the Kabat numbering.
  • the hapten is bound in a deep pocket formed by hydrophobic residues.
  • a fluorescent digoxigenin-Cy5 conjugate was used in this crystallographic study, wherein the fluorophore as well as the linker between digoxigenin and Cy5 were not visible in the structure due to a high flexibility and resulting disorder in the crystal.
  • the linker and Cy5 are attached to 032 of digoxigenin which points into the direction of the CDR2 of the heavy chain.
  • the distance between 032 of digoxigenin to the Ca of the amino acid residue in position 52b according to Kabat is about 10.5 ⁇ .
  • this position is a “universal” position, i.e. the position is applicable to any (anti-hapten) antibody and, thus, it is not required to start from scratch every time a new (anti-hapten) antibody has to be modified by providing the crystal structure and determining the appropriate position that enables hapten-positioned covalent coupling.
  • VH52bC or VH53C, respectively, according to the numbering scheme of Kabat could unexpectedly be used for each hapten-binding antibody analyzed. Even though the antibodies and structures of their binding pockets are quite diverse, it has been shown that the VH52bC/VH53C mutation can be used for covalent attachment of haptens/haptenylated compounds to antibodies that bind the hapten (such as e.g. digoxigenin, biotin, fluorescein, theophylline as well as bromodeoxyuridine).
  • haptens/haptenylated compounds such as e.g. digoxigenin, biotin, fluorescein, theophylline as well as bromodeoxyuridine.
  • the antibodies modified as reported herein retain the hapten binding capability of their parent (i.e. wild-type) anti-hapten antibody.
  • the anti-hapten antibody comprising an artificial antibody cysteine residue binds, in one embodiment specifically binds to a hapten.
  • an antibody that binds to denote that the antibody can form a complex with its antigen in a specific manner.
  • the specific binding can be detected in an in vitro assay, such as in a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden).
  • the affinity of the complex formation is defined by the terms k a (rate constant for the association of the compounds to form the complex), k D (dissociation constant, dissociation of the complex), and K D (k D /ka).
  • Binding or specifically binding means a binding affinity (K D ) of about 10 ⁇ 8 M or less, in one embodiment of about 10 ⁇ 8 M to about 10 ⁇ 13 M, in one embodiment of about 10 ⁇ 9 M to about 10 ⁇ 13 M.
  • an antibody that binds to a hapten to form a complex as reported herein specifically binds to the hapten with a binding affinity (K D ) of about 10 ⁇ 8 mol/1 or less, in one embodiment of about 10 ⁇ 8 mol/1 to about 10 ⁇ 13 mol/1, in one embodiment of about 10 ⁇ 9 mol/1 to 10 ⁇ 13 mol/l.
  • the disulfide bridge between the two compounds is formed spontaneously upon formation of the non-covalent complex. Therefore, a method for the formation of a covalent complex as reported herein simply requires the mixing of the two compounds. The only pre-requisite for the formation of the disulfide bond is a proper orientation of the two compounds with respect to each other.
  • the artificial antibody cysteine residue containing anti-hapten antibodies may be site-specifically and efficiently coupled with a haptenylated polypeptide comprising an artificial polypeptide cysteine residue.
  • the anti-digoxigenin antibody is characterized in comprising at least one, two, three, four, five, or six CDRs selected from (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 09 or 25, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10 or 26, (c) heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 27, (d) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or 29, (e) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or 30, and (f) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or 31.
  • CDRs selected from (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 09 or 25, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10 or 26, (c) heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 27, (d) light chain
  • the anti-biotin antibody is characterized in comprising at least one, two, three, four, five, or six CDRs selected from (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 41 or 57, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42 or 58, (c) heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 43 or 59, (d) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 45 or 61, (e) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46 or 62, and (f) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 47 or 64.
  • CDRs selected from (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 41 or 57, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42 or 58, (c) heavy chain CDR3 comprising the amino acid sequence of
  • the anti-fluorescein antibody is characterized in comprising at least one, two, three, four, five, or six CDRs selected from (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 105 or 113, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 106 or 114, (c) heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 107 or 115, (d) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 109 or 117, (e) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110 or 118, and (f) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 111 or 119.
  • the anti-theophylline antibody is characterized in comprising at least one, two, three, four, five, or six CDRs selected from (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 73 or 89, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 74 or 90, (c) heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 75 or 91, (d) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 77 or 93, (e) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 78 or 94, and (f) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 79 or 95.
  • CDRs selected from (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 73 or 89, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 74 or 90, (c) heavy chain CDR3
  • the anti-digoxigenin antibody is characterized in comprising (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 09 or 25, (ii) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10 or 26, and (iii) heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 11 or 27, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or 29, (ii) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or 30, and (c) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or 31.
  • the anti-biotin antibody is characterized in comprising (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 41 or 57, (ii) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42 or 58, and (iii) heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 43 or 59, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 45 or 61, (ii) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46 or 6242, and (c) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 47 or 63.
  • the anti-fluorescein antibody is characterized in comprising (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 105 or 113, (ii) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 106 or 114, and (iii) heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 107 or 115, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 109 or 117, (ii) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110 or 118, and (c) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 111 or 119.
  • the anti-theophylline antibody is characterized in comprising (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 73 or 89, (ii) heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 74 or 90, and (iii) heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 75 or 91, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 77 or 93, (ii) light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 78 or 94, and (c) light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 79 or 95.
  • the anti-digoxigenin antibody, and/or the anti-biotin antibody, and/or the anti-theophylline antibody is a humanized antibody.
  • the anti-digoxigenin antibody comprises CDRs as in any of the above embodiments and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.
  • acceptor human framework e.g. a human immunoglobulin framework or a human consensus framework.
  • the anti-biotin antibody comprises CDRs as in any of the above embodiments and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.
  • acceptor human framework e.g. a human immunoglobulin framework or a human consensus framework.
  • the anti-theophylline antibody comprises CDRs as in any of the above embodiments and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.
  • acceptor human framework e.g. a human immunoglobulin framework or a human consensus framework.
  • the anti-digoxigenin antibody is characterized in comprising a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 04 or 12 or 20 or 28.
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-digoxigenin antibody comprising that sequence retains the ability to bind to digoxigenin.
  • the anti-digoxigenin antibody comprises the VH sequence in SEQ ID NO: 01 or 09 or 17 or 25, including post-translational modifications of that sequence.
  • the anti-digoxigenin antibody is characterized in comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 08 or 16 or 24 or 32.
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-digoxigenin antibody comprising that sequence retains the ability to bind to digoxigenin.
  • the anti-digoxigenin antibody comprises the VL sequence in SEQ ID NO: 08 or 16 or 24 or 32, including post-translational modifications of that sequence.
  • the anti-biotin antibody is characterized in comprising a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 44 or 52 or 60.
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-biotin antibody comprising that sequence retains the ability to bind to biotin.
  • the anti-biotin antibody comprises the VH sequence in SEQ ID NO: 36 or 44 or 52 or 60, including post-translational modifications of that sequence.
  • the anti-fluorescein antibody is characterized in comprising a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 108 or 116.
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-fluorescein antibody comprising that sequence retains the ability to bind to fluorescein.
  • the anti-fluorescein antibody comprises the VH sequence in SEQ ID NO: 108 or 116, including post-translational modifications of that sequence.
  • the anti-theophylline antibody is characterized in comprising a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 68 or 76 or 84 or 92.
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-theophylline antibody comprising that sequence retains the ability to bind to theophylline.
  • the anti-theophylline antibody comprises the VH sequence in SEQ ID NO: 68 or 76 or 84 or 92 including post-translational modifications of that sequence.
  • the anti-digoxigenin antibody is characterized in comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 04 or 12 or 20 or 28, and SEQ ID NO: 08 or 16 or 24 or 32, respectively, including post-translational modifications of those sequences.
  • the anti-biotin antibody is characterized in comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 36 or 44 or 52 or 60, and SEQ ID NO: 40 or 48 or 56 or 64, respectively, including post-translational modifications of those sequences.
  • the anti-fluorescein antibody is characterized in comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 108 or 116, and SEQ ID NO: 112 or 120, respectively, including post-translational modifications of those sequences.
  • the anti-theophylline antibody is characterized in comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 68 or 76 or 84 or 92, and SEQ ID NO: 72 or 80 or 88 or 96, respectively, including post-translational modifications of those sequences.
  • a further position that was identified as modification point is the position VH28 according to the Kabat numbering.
  • ESI-MS analyses demonstrate that covalent antibody conjugation of haptenylated therapeutic peptides result in a conjugate of defined size which is larger than non-complexed antibody or non-complexed peptide.
  • the antibody as reported herein itself or the antibody in the complex as reported herein has a dissociation constant (Kd) of ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. of about 10 ⁇ 8 M or less, e.g. from about 10 ⁇ 8 M to about 10 ⁇ 13 M, e.g., from about 10 ⁇ 9 M to about 10 ⁇ 13 M).
  • Kd dissociation constant
  • Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay.
  • Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881).
  • MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.).
  • a non-adsorbent plate (Nunc #269620)
  • 100 pM or 26 pM [ 125 I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta, L. G.
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 ⁇ l/well of scintillant (MICROSCINT-20TM; Packard) is added, and the plates are counted on a TOPCOUNTTM gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.
  • Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CMS chips at ⁇ 10 response units (RU).
  • CMS carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml (about 0.2 ⁇ M) before injection at a flow rate of 5 ⁇ l/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block non-reacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25° C. at a flow rate of approximately 25 ⁇ l/min.
  • TWEEN-20TM polysorbate 20
  • association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio k off /k on . See, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • recombinant host cells e.g. E. coli or phage
  • the antibody in a conjugate as reported herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L. G. et al., J. Immunol.
  • the antibody in a conjugate as reported herein is a human antibody.
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk, M. A. and van de Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes.
  • the endogenous immunoglobulin loci have generally been inactivated.
  • Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95) Human antibodies generated via human B-cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
  • Antibodies in the conjugate as reported herein may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods in Molecular Biology 178 (2001) 1-37 and further described, e.g., in the McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol. Biol.
  • repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455.
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths, A. D. et al., EMBO J. 12 (1993) 725-734.
  • naive libraries can also be made synthetically by cloning non-rearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol. Biol. 227 (1992) 381-388.
  • Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.
  • Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
  • one or more scFv antibody fragments can be fused to the C-terminus of one or more polypeptide chains of a complete antibody. Especially to each heavy chain C-terminus or to each light chain C-terminus a scFv antibody fragment can be fused.
  • one or more antibody Fab fragments can be fused to the C-terminus of one or more polypeptide chains of a complete antibody. Especially to each heavy chain C-terminus or to each light chain C-terminus an antibody Fab fragment can be fused.
  • one scFv and one antibody Fab fragment can be fused to the N-termini of an antibody Fc-region.
  • one scFv or antibody Fab fragment can be fused to an N-terminus of an antibody Fc-region and one scFv or antibody Fab fragment can be fused to the C-terminus of the respective other chain of an antibody Fc-region.
  • a wide variety of recombinant antibody formats have been developed, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech 25 (2007) 1233-1234).
  • All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N. and Leger, O., Pathobiology 74 (2007) 3-14). It has to be kept in mind that one may want to retain effector functions, such as e.g. complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), which are mediated through the Fc receptor binding, by maintaining a high degree of similarity to naturally occurring antibodies.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody dependent cellular cytotoxicity
  • WO 2007/024715 are reported dual variable domain immunoglobulins as engineered multivalent and multispecific binding proteins.
  • a process for the preparation of biologically active antibody dimers is reported in U.S. Pat. No. 6,897,044.
  • Multivalent Fv antibody construct having at least four variable domains which are linked with each over via peptide linkers are reported in U.S. Pat. No. 7,129,330.
  • Dimeric and multimeric antigen binding structures are reported in US 2005/0079170.
  • Tri- or tetra-valent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other covalently by a connecting structure, which protein is not a natural immunoglobulin are reported in U.S. Pat. No. 6,511,663.
  • bispecific antibodies are reported that can be efficiently expressed in prokaryotic and eukaryotic cells, and are useful in therapeutic and diagnostic methods.
  • a method of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers is reported in US 2005/0163782.
  • Bispecific tetravalent receptors are reported in U.S. Pat. No. 5,959,083.
  • Engineered antibodies with three or more functional antigen binding sites are reported in WO 2001/077342.
  • Multispecific and multivalent antigen-binding polypeptides are reported in WO 1997/001580.
  • WO 1992/004053 reports homoconjugates, typically prepared from monoclonal antibodies of the IgG class which bind to the same antigenic determinant are covalently linked by synthetic cross-linking.
  • Oligomeric monoclonal antibodies with high avidity for antigen are reported in WO 1991/06305 whereby the oligomers, typically of the IgG class, are secreted having two or more immunoglobulin monomers associated together to form tetravalent or hexavalent IgG molecules.
  • Sheep-derived antibodies and engineered antibody constructs are reported in U.S. Pat. No.
  • an antibody provided herein or the antibody in a conjugate as reported herein is a multispecific antibody, e.g. a bispecific antibody.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for a hapten and the other is for any other (non-hapten) antigen.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168).
  • Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody” technology for making bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad.
  • the CH3 domains of the heavy chains of the bispecific antibody are altered by the “knob-into-holes” technology which is described in detail with several examples in e.g. WO 96/027011, WO 98/050431, Ridgway J. B., et al., Protein Eng. 9 (1996) 617-621, Merchant, A. M., et al., Nat Biotechnol 16 (1998) 677-681.
  • the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing these two CH3 domains.
  • Each of the two CH3 domains (of the two heavy chains) can be the “knob”, while the other is the “hole”.
  • the antibodies as reported herein are in one embodiment characterized in that
  • the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophane (W).
  • amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).
  • both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.
  • C cysteine
  • the bispecific antibody comprises a T366W mutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the “hole chain”.
  • An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A. M, et al., Nature Biotech 16 (1998) 677-681) e.g.
  • the bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains.
  • the bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming a interchain disulfide bridge) (numbering according to EU index of Kabat; (Kabat, E.
  • the bispecific antibody comprises a T366W mutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the “hole chain” and additionally R409D, K370E mutations in the CH3 domain of the “knobs chain” and D399K, E357K mutations in the CH3 domain of the “hole chain”.
  • the bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or the bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains and additionally R409D, K370E mutations in the CH3 domain of the “knobs chain” and D399K, E357K mutations in the CH3 domain of the “hole chain”.
  • Such knob and hole mutations in the CH3 domain are typically used in human heavy chain constant regions of SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, or SEQ ID NO: 172 (human IgG1 subclass allotypes (Caucasian and Afro-American or mutants L234A/L235A, and L234A/L235A/P329G), SEQ ID NO: 173, SEQ ID NO: 174, or SEQ ID NO: 175 (human IgG4 subclass or mutants S228P, L235E, and S228P/L235E/P329G) (numbering according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
  • the bispecific antibody comprises human heavy chain constant regions of SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, or SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, or SEQ ID NO: 175 further including such “knob” and “hole” mutations in the CH3 domain (e.g.
  • the antibody or fragment herein also includes a “Dual Acting Fab” or “DAF” comprising an antigen binding site that binds to a hapten as well as another, different antigen (see US 2008/0069820, for example).
  • the antibody or fragment herein also includes multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793.
  • the first binding specificity of the bispecific antibody is to a hapten and the second binding specificity is to a non-hapten antigen.
  • the non-hapten antigen is selected from the leukocyte markers, CD2, CD3, CD4, CDS, CD6, CD7, CD8, CD11a,b,c, CD13, CD14, CD18, CD19, CD22, CD23, CD27 and its ligand, CD28 and its ligands B7.1, B7.2, B7.3, CD29 and its ligand, CD30 and its ligand, CD40 and its ligand gp39, CD44, CD45 and isoforms, CD56, CD58, CD69, CD72, CTLA-4, LFA-1 and TCR; the histocompatibility antigens, MHC class I or II, the Lewis Y antigens, SLex, SLey, SLea, and SLeb; the integrins, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5
  • amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in HVRs, e.g., to improve antibody affinity.
  • Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, P. S., Methods Mol. Biol. 207 (2008) 179-196), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom, H. R. et al.
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. Heavy chain CDR3 and light chain CDR3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR “hotspots” or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science 244 (1989) 1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody provided herein or comprised in a conjugate as reported herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc-region. See, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997) 26-32.
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc-region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc-region (EU numbering of Fc-region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621.
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N.
  • Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003/0157108; and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).
  • Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc-region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc-region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.
  • one or more amino acid modifications may be introduced into the Fc-region of an antibody provided herein, thereby generating an Fc-region variant.
  • the Fc-region variant may comprise a human Fc-region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc-region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc(RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, J. V. and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492.
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al., Proc.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656.
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood 101 (2003) 1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103 (2004) 2738-2743).
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int. Immunol. 18 (2006: 1759-1769).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc-region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
  • an antibody variant comprises an Fc-region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc-region (EU numbering of residues).
  • alterations are made in the Fc-region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164 (2000) 4178-4184.
  • CDC Complement Dependent Cytotoxicity
  • Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US 2005/0014934.
  • Those antibodies comprise an Fc-region with one or more substitutions therein which improve binding of the Fc-region to FcRn.
  • Such Fc variants include those with substitutions at one or more of Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc-region residue 434 (U.S. Pat. No. 7,371,826).
  • cysteine engineered antibodies e.g., “thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc-region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
  • an antibody provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., g
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or non-branched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • conjugates of an antibody and non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the non-proteinaceous moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605).
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed.
  • Payload denotes any molecule or combination of molecules whose activity it is desired to be delivered (in)to and/or localize at a cell. Payloads include, but are not limited to labels, polypeptide toxins (e.g. Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and the like), enzymes, growth factors, transcription factors, drugs, radionuclides, ligands, antibodies, liposomes, nanoparticles, viral particles, cytokines, and the like.
  • polypeptide toxins e.g. Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and the like
  • enzymes e.g. Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and the like
  • growth factors e.g. Pseudomonas exotoxin, ricin, abrin
  • the haptenylated polypeptide as reported herein may be further conjugated, if the polypeptide is not by itself one of the molecules, to a therapeutic agent (drug), a polypeptide toxin (e.g. a toxin such as doxorubicin or pertussis toxin), a fluorophore such as a fluorescent dye like fluorescein or rhodamine, a chelating agent for an imaging or radiotherapeutic metal, a peptidyl or non-peptidyl label or detection tag, or a clearance-modifying agent such as various isomers of polyethylene glycol, a peptide that binds to a third component, or another carbohydrate or lipophilic agent.
  • the conjugation can be either directly or via an intervening linker.
  • the drug moiety (D) of the hapten-drug conjugate (ADC, haptenylated drug) can be any compound, moiety or group which has a cytotoxic or cytostatic effect.
  • Drug moieties include: (i) chemotherapeutic agents, which may function as microtubule inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators; (ii) polypeptide toxins, which may function enzymatically; and (iii) radioisotopes.
  • Exemplary drug moieties include, but are not limited to, a maytansinoid, an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicin and other enediyne antibiotics, a taxane, an anthracycline, and stereoisomers, isosters, analogs or derivatives thereof.
  • Polypeptide toxins include diphtheria-A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain (Vitetta et al (1987) Science, 238:1098), abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-5), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes (WO 93/21232).
  • Therapeutic radioisotopes include 32P, 33P, 90Y, 125I, 131I, 131In, 153Sm, 186Re, 188Re, 211At, 212B, 212Pb, and radioactive isotopes of Lu.
  • radioisotope or other labels may be incorporated in known ways (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57; “Monoclonal Antibodies in Immunoscintigraphy” Chatal, CRC Press 1989).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of a radionuclide to the complex (WO 94/11026).
  • the haptenylated polypeptide can further comprise a haptenylated label.
  • Any label moiety which can be covalently attached to the hapten can be used (see e.g. Singh et al (2002) Anal. Biochem. 304:147-15; Harlow E. and Lane, D. (1999) Using Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Lundblad R. L. (1991) Chemical Reagents for Protein Modification, 2nd ed. CRC Press, Boca Raton, Fla.).
  • the label may function to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g.
  • FRET fluorescence resonance energy transfer
  • mobility e.g. electrophoretic mobility or cell-permeability, by charge, hydrophobicity, shape, or other physical parameters
  • a capture moiety e.g. to modulate ionic complexation
  • Conjugates comprising a haptenylated polypeptide and a label as reported herein may be useful in diagnostic assays, e.g., for detecting expression of an antigen of interest in specific cells, tissues, or serum.
  • a bispecific antibody will be used wherein the first binding specificity binds to a target and the second binding specificity binds to a haptenylated label.
  • the haptenylated polypeptide will typically be labeled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories:
  • Radioisotopes radioisotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131Bi. Radioisotope labeled conjugates are useful in receptor targeted imaging experiments.
  • the haptenylated polypeptide can be further labeled with ligand reagents that bind, chelate or otherwise complex a radioisotope metal using the techniques described in Current Protocols in Immunology, (1991) Volumes 1 and 2, Coligen et al, Ed.
  • Chelating ligands which may complex a metal ion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.).
  • Radionuclides can be targeted via complexation with the complex as reported herein (Wu et al, Nature Biotechnology 23(9) (2005) 1137-1146).
  • Receptor target imaging with radionuclide labeled complexes can provide a marker of pathway activation by detection and quantification of progressive accumulation of complexes or corresponding therapeutic antibodies in tumor tissue (Albert et al (1998) Bioorg. Med. Chem. Lett. 8:1207-1210).
  • Metal-chelate complexes suitable as labels for imaging experiments (US 2010/0111856; U.S. Pat. Nos. 5,342,606; 5,428,155; 5,316,757; 5,480,990; 5,462,725; 5,428,139; 5,385,893; 5,739,294; 5,750,660; 5,834,456; Hnatowich et al, J. Immunol. Methods 65 (1983) 147-157; Meares et al, Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al, Bioconjugate Chem. 1 (1990) 59-65; Meares et al, J. Cancer (1990), Suppl.
  • Fluorescent labels such as rare earth chelates (europium chelates), fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; rhodamine types including TAMRA; dansyl; Lissamine; cyanines; phycoerythrins; Texas Red; and analogs thereof.
  • the fluorescent labels can be conjugated to the polypeptide using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescent dyes and fluorescent label reagents include those which are commercially available from Invitrogen/Molecular Probes (Eugene, Oreg., USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).
  • Detection labels such as fluorescent dyes and chemiluminescent dyes (Briggs et al “Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally applicable for labeling, especially with the following properties: (i) the labeled conjugate should produce a very high signal with low background so that small quantities of conjugate can be sensitively detected in both cell-free and cell-based assays; and (ii) the labeled conjugate should be photostable so that the fluorescent signal may be observed, monitored and recorded without significant photo bleaching.
  • the labels should (iii) have good water-solubility to achieve effective conjugate concentration and detection sensitivity and (iv) are non-toxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.
  • the enzyme generally catalyzes a chemical alteration of a chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • luciferases e.g., firefly luciferase and bacterial lucifer
  • enzyme-substrate combinations include, for example:
  • HRP Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3′,5,5′-tetramethylbenzidine hydrochloride
  • the labeled haptenylated polypeptide as reported herein may be employed in any known assay method, such as ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques (1987) pp. 147-158, CRC Press, Inc.).
  • Labeled haptenylated polypeptides as reported herein are useful as imaging biomarkers and probes by the various methods and techniques of biomedical and molecular imaging such as: (i) MRI (magnetic resonance imaging); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Tinianow, J. et al Nuclear Medicine and Biology, 37(3) (2010) 289-297; Chen et al, Bioconjugate Chem. 15 (2004) 41-49; US 2010/0111856 (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound.
  • MRI magnetic resonance imaging
  • MicroCT computerized tomography
  • SPECT single photon emission computed tomography
  • PET positron emission tomography
  • Imaging biomarkers may be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.
  • Biomarkers may be of several types: Type 0 markers are natural history markers of a disease and correlate longitudinally with known clinical indices, e.g.
  • Imaging biomarkers thus can provide pharmacodynamic (PD) therapeutic information about: (i) expression of a target protein, (ii) binding of a therapeutic to the target protein, i.e. selectivity, and (iii) clearance and half-life pharmacokinetic data.
  • PD pharmacodynamic
  • in vivo imaging biomarkers relative to lab-based biomarkers include: non-invasive treatment, quantifiable, whole body assessment, repetitive dosing and assessment, i.e. multiple time points, and potentially transferable effects from preclinical (small animal) to clinical (human) results. For some applications, bioimaging supplants or minimizes the number of animal experiments in preclinical studies.
  • the antibody in a conjugate as reported herein may be further conjugated, if it is not by itself one of the molecules, to a therapeutic agent (drug), a polypeptide toxin (e.g. a toxin such as doxorubicin or pertussis toxin), a fluorophores such as a fluorescent dye like fluorescein or rhodamine, a chelating agent for an imaging or radiotherapeutic metal, a peptidyl or non-peptidyl label or detection tag, or a clearance-modifying agent such as various isomers of polyethylene glycol, a peptide that binds to a third component, or another carbohydrate or lipophilic agent.
  • a therapeutic agent drug
  • a polypeptide toxin e.g. a toxin such as doxorubicin or pertussis toxin
  • a fluorophores such as a fluorescent dye like fluorescein or rhodamine
  • the invention also provides immunoconjugates comprising an antibody as reported herein or a conjugate as reported herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and EP 0 425 235 B1); an auristatin such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos.
  • ADC antibody-drug conjugate
  • drugs including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and EP 0 425 235 B1); an auristatin such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF)
  • an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • an enzymatically active toxin or fragment thereof including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain
  • an immunoconjugate comprises an antibody as described herein or a complex as reported herein conjugated to a radioactive atom to form a radioconjugate.
  • a variety of radioactive isotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the radioconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example TC 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • Conjugates of an antibody and a cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (
  • a ricin immunotoxin can be prepared as described in Vitetta, E. S. et al., Science 238 (1987) 1098-1104.
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.
  • the linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari, R. V. et al., Cancer Res. 52 (1992) 127-131; U.S. Pat. No. 5,208,020) may be used.
  • the immunoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).
  • cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC,
  • the hapten in a conjugate as reported herein is in one embodiment conjugated to a cytotoxic agent, such as e.g. a toxin such as doxorubicin or pertussis toxin.
  • a cytotoxic agent such as e.g. a toxin such as doxorubicin or pertussis toxin.
  • a conjugate is denoted as haptenylated polypeptide toxin.
  • the conjugation can be either directly or via an intervening linker.
  • the polypeptide toxin is in one embodiment a protein toxin, which may function enzymatically.
  • Protein toxins include diphtheria-A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain (Vitetta et al (1987) Science, 238:1098), abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-5), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes (WO 93/21232).
  • Conjugates between the hapten and the polypeptide toxin may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluor
  • a ricin immunotoxin can be prepared as described in Vitetta, E. S. et al., Science 238 (1987) 1098-1104.
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.
  • the linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari, R. V. et al., Cancer Res. 52 (1992) 127-131; U.S. Pat. No. 5,208,020) may be used.
  • the haptenylated polypeptide toxin is made using cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).
  • cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA
  • linker denotes a bifunctional or multifunctional moiety which can be used to conjugate (link) the hapten to the polypeptide.
  • Haptenylated polypeptides can be conveniently prepared using a linker having reactive functionality for binding to the polypeptide and the hapten.
  • a linker has a reactive site which has an electrophilic group that is reactive to a nucleophilic group present on the polypeptide.
  • a cysteine thiol group for example is reactive with an electrophilic group on a linker and forms a covalent bond to a linker.
  • Useful electrophilic groups include, but are not limited to, another thiol, maleimide and haloacetamide groups (see e.g. conjugation method at page 766 of Klussman et al, Bioconjugate Chemistry 15(4) (2004) 765-773).
  • thiol-reaction functional groups include, but are not limited to, thiol, maleimide, alpha-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.
  • the linker may comprise amino acid residues which link the hapten to the polypeptide.
  • the amino acid residues may form a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
  • Amino acid residues include those occurring naturally, as well as non-naturally occurring amino acid analogs, such as e.g. citrulline or ⁇ -amino acids, such as e.g. ⁇ -alanine, or ⁇ -amino acids such as 4-amino-butyric acid.
  • the linker has a reactive functional group which has a nucleophilic group that is reactive to an electrophilic group present on the hapten.
  • Useful electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups.
  • the heteroatom of a nucleophilic group of a linker can react with an electrophilic group on the hapten or the polypeptide and form a covalent bond to the hapten or the polypeptide.
  • Useful nucleophilic groups on a linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • the electrophilic group on the hapten provides a convenient site for attachment to a linker.
  • peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments.
  • Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (E. Schroder and K. Lubke “The Peptides”, volume 1 (1965) 76-136, Academic Press) which is well known in the field of peptide chemistry.
  • the linker may be substituted with groups which modulated solubility or reactivity.
  • a charged substituent such as sulfonate (50 3 ⁇ ) or ammonium or a polymer such as PEG, may increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the hapten or the polypeptide, or facilitate the coupling reaction depending on the synthetic route employed.
  • conjugates comprising a hapten an a polypeptide as reported herein expressly contemplate, but are not limited to, complexes prepared with linker reagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone) benzoate), and including bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO) 3 , and BM(PEO) 4 , which are commercially available from Pierce Biotechnology, Inc.
  • Bis-maleimide reagents allow the attachment of e.g. a thiol group to a thiol-containing drug moiety, label, or linker intermediate, in a sequential or concurrent fashion.
  • Other functional groups besides maleimide, which are reactive with e.g. a thiol group include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.
  • Exemplary linker include a valine-citrulline (val-cit or vc) dipeptide linker reagent having a maleimide stretcher and a para-aminobenzylcarbamoyl (PAB) self-immolative spacer, and a phe-lys(Mtr) dipeptide linker reagent having a maleimide Stretcher unit and a p-amino benzyl self-immolative spacer.
  • valine-citrulline val-cit or vc
  • PAB para-aminobenzylcarbamoyl
  • Cysteine thiol groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker reagents and haptenylated compounds including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups; and (iv) disulfides, including pyridyl disulfides, via sulfide exchange.
  • active esters such as NHS esters, HOBt esters, haloformates, and acid halides
  • alkyl and benzyl halides such as haloacetamides
  • aldehydes ketones, carboxyl, and maleimide groups
  • disulfides including pyridyl disulfides, via sulfide exchange.
  • Nucleophilic groups on a haptenylated compound include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents.
  • the DNA encoding the amino acid sequence variant of the antibody as reported herein or as comprised in a conjugate as reported herein can be prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may be constructed also by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. Mutagenic primers encode the cysteine codon replacement(s). Standard mutagenesis techniques can be employed to generate DNA encoding such modified engineered antibodies.
  • Polypeptides and antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567.
  • isolated nucleic acid encoding the polypeptide and an antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody) or the amino acid sequence of the polypeptide.
  • one or more vectors comprising such nucleic acid are provided.
  • a host cell comprising such nucleic acid is provided.
  • a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g.
  • a method of making an antibody as reported herein comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254, describing expression of antibody fragments in E. coli .)
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J. P., Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ⁇ CHO cells (Urlaub, G. et al., Proc. Natl.
  • compositions comprising the conjugate as reported herein are prepared by mixing such conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arg
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rhuPH20 HYLENEX®, Baxter International, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in US 2005/0260186 and US 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958.
  • Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulations including a histidine-acetate buffer.
  • the formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody or conjugate, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • a conjugate as reported herein for use as a medicament is provided.
  • a conjugate as reported herein for use in treating a disease is provided.
  • a conjugate as reported herein for use in a method of treatment is provided.
  • the invention provides a conjugate as reported herein for use in a method of treating an individual comprising administering to the individual an effective amount of the conjugate as reported herein.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
  • An “individual” according to any of the above embodiments may be a human.
  • the invention provides for the use of a conjugate as reported herein in the manufacture or preparation of a medicament.
  • the medicament is for treatment of a disease.
  • the medicament is for use in a method of treating a disease comprising administering to an individual having a disease an effective amount of the medicament.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
  • An “individual” according to any of the above embodiments may be a human.
  • the invention provides a method for treating a disease.
  • the method comprises administering to an individual having such a disease an effective amount of a conjugate as reported herein.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.
  • An “individual” according to any of the above embodiments may be a human.
  • the invention provides pharmaceutical formulations comprising any of the conjugates as reported herein, e.g., for use in any of the above therapeutic methods.
  • a pharmaceutical formulation comprises any of the conjugates as reported herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprises any of the conjugates as reported herein and at least one additional therapeutic agent, e.g., as described below.
  • Conjugates as reported herein can be used either alone or in combination with other agents in a therapy.
  • a conjugate as reported herein may be co-administered with at least one additional therapeutic agent.
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • Conjugates as reported herein can also be used in combination with radiation therapy.
  • a conjugate as reported herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Conjugates as reported herein would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the conjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • the appropriate dosage of a conjugate as reported herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of conjugate, the severity and course of the disease, whether the conjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the conjugate, and the discretion of the attending physician.
  • the conjugate is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) of conjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the conjugate would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the conjugate).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an antibody or a complex as reported herein.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody or a complex as reported herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • Ringer's solution such as phosphate
  • Fab fragments were generated by protease digestion of the purified IgGs and subsequently purified, applying well known state of the art methods (papain digestion).
  • Crystallization of the apo Fab fragment (purified Fabs) in 20 mM His-HCl 140 mM NaCl, pH 6.0 were concentrated to 13 mg/ml. Crystallization droplets were set up at 21° C. by mixing 0.2 ⁇ l of protein solution with 0.2 ⁇ L reservoir solution in vapor diffusion sitting drop experiments. Crystals appeared out of 0.1 M Tris pH 8.5, 0.01 M cobalt chloride, 20% polyvinylpyrrolidone K15 within 5 days and grew to a final size of 0.3 mm ⁇ 0.06 mm ⁇ 0.03 mm within 8 days.
  • Crystals were harvested with 15% Glycerol as cryoprotectant and then flash frozen in liquid N2. Diffraction images were collected with a Pilatus 6M detector at a temperature of 100K at the beam line X10SA of the Swiss Light Source and processed with the programs XDS (Kabsch, W., J. Appl. Cryst. 26 (1993) 795-800) and scaled with SCALA (obtained from BRUKER AXS), yielding data to 2.22 ⁇ resolution.
  • Standard crystallographic programs from the CCP4 software suite were used to solve the structure by molecular replacement with the PDB entry 3PQP as search model, to calculate the electron density, and to refine the x-ray structure (CCP4, Collaborative Computational Project, Acta Crystallogr. D, 760-763 (1994)).
  • the structural models were rebuilt into the electron density using COOT (Emsley, P., et al. Acta Crystallogr. D Biol. Crystallogr. 60 (2010) 486-501). Coordinates were refined with REFMACS (Murshudov, G. N., et al. Acta Crystallogr. D Biol. Crystallogr. 53 (1997) 240-55) and with autoBUSTER (Global Phasing Ltd.).
  • apo Crystals of the Fab fragment used for soaking experiments were derived out of 0.8 M succinic acid within 3 days after screening and grew to a final size of 0.25 mm ⁇ 0.04 mm ⁇ 0.04 mm within 5 days.
  • Biocytinamid was dissolved at 100 mM in water. Subsequently, the compound was diluted to 10 mM working concentration in crystallization solution and applied to the crystals in the crystallization droplet.
  • Crystals were washed three times with 2 ⁇ l of 10 mM compound solution and were finally incubated for 16 h with biocytinamid at 21° C.
  • Crystals were harvested with 15% glycerol as cryoprotectant and then flash frozen in liquid N 2 .
  • Diffraction images were collected with a Pilatus 6M detector at a temperature of 100 K at the beam line X10SA of the Swiss Light Source and processed with the programs XDS (Kabsch, W., J. Appl. Cryst. 26 (1993) 795-800) and scaled with SCALA (obtained from BRUKER AXS), yielding data to 2.35 ⁇ resolution.
  • Standard crystallographic programs from the CCP4 software suite were used to solve the structure by molecular replacement with the coordinates of the apo Fab fragment as search model, to calculate the electron density, and to refine the x-ray structure to a resolution of 2.5 ⁇ (CCP4).
  • the structural models were rebuilt into the electron density using COOT Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of COOT. Acta Crystallogr. D Biol. Crystallogr. 60, 486-501 (2010)). Coordinates were refined with REFMACS (Murshudov, G. N., et al. Acta Crystallogr. D Biol. Crystallogr. 53, 240-255 (1997)) and with autoBUSTER (Global Phasing Ltd.).
  • the result of the experimental structure determination is shown in FIG. 6 .
  • the crystal form of the complex contained four independent biocytinamid:anti-biotin Fab complexes in the asymmetric unit, with biocytinamid bound similarly by all Fab molecules.
  • Biocytinamid is bound in a pocket formed by CDRs 1 and 3 of the heavy chain and all 3 light chain CDRs.
  • the binding pocket of the ligand is defined by residues ASN29, ASP31, THR32, PHE33, GLN35, TRP99 and TRP106 from the heavy chain and ASN31, TYR32, LEU33, SER34, TYR49, SER50, PHE91 and TYR96 from the light chain.
  • the biotin head group forms hydrogen bonds with residues of CDR2 and CDR1 at one end of the pocket: N3 of biocytinamid is interacting with the hydroxyl-oxygen of Ser50 whereas O22 is in contact with the backbone-amide nitrogen of the same residue.
  • O22 of biocytinamid is also hydrogen-bonded to the hydroxyl-group oxygen of Ser34.
  • hydrophobic interactions are observed between biocytinamid and the aromatic side chains lining the binding pocket.
  • the amide bond at the end of the (CH 2 ) 4 aliphatic tail of biotin stacks onto PHE33 of heavy chain CDR1 and is stabilized by an additional hydrogen bond to the backbone amide nitrogen of PHE33 and to Asp31. This positions the amide nitrogen, which is the site of linkage to the active entity, in a way that atoms that are following the nitrogen are pointing away from the binding pocket towards the solvent.
  • the positions to be mutated must simultaneously meet two requirements: (i) the coupling positions should be in proximity to the binding region to utilize the antigen/hapten positioning effect for directed coupling, and (ii) the mutation and coupling position must be positioned in a manner that antigen/hapten binding by itself is not affected.
  • These requirements for finding a suitable position are de facto ‘contradicting’ each other because requirement (i) is best served by a position close to the binding site, while requirement (ii) is most safely achieved by positions that are distant from the binding site.
  • the first position is located at position VH52b or VH53 according to the Kabat numbering depending on the actual length of the CDR2 of the respective antibody.
  • the hapten is bound in a deep pocket formed by hydrophobic residues.
  • a fluorescent digoxigenin-Cy5 conjugate was used in this crystallographic study, wherein the fluorophore as well as the linker between digoxigenin and Cy5 were not visible in the structure due to a high flexibility and resulting disorder in the crystal.
  • the linker and Cy5 are attached to 032 of digoxigenin which points into the direction of the CDR2 of the heavy chain.
  • the distance between 032 (see above) of digoxigenin to the Ca of the amino acid residue in position 52b according to Kabat numbering is 10.5 ⁇ .
  • a further position that was identified as modification point is the position VH28 according to the Kabat numbering.
  • one of these positions is a ‘universal’ position, i.e. this position is applicable to any antibody and, thus, it is not required to start from scratch every time a new antibody has to be modified by providing the crystal structure and determining the appropriate position that enables hapten-positioned covalent coupling.
  • VH52bC or VH53C, respectively, according to Kabat heavy chain variable region numbering could be used for each hapten-binding antibody (anti-hapten antibody). Even though the antibodies and structures of their binding pockets are quite diverse, it has been shown that the VH52bC/VH53C mutation can be used for covalent attachment of antigens/haptens to antibodies that bind digoxigenin, biotin, fluorescein, as well as theophylline.
  • Binding entities that are composed of these sequences could be expressed and purified with standard Protein A- and size exclusion chromatography (see Example 3).
  • the resulting molecules were fully functional and retained affinity towards their cognate haptens in the same manner as their unmodified parent molecules. This was demonstrated by Surface-Plasmon-Resonance (SPR) experiments (see Example 4).
  • SPR Surface-Plasmon-Resonance
  • Murine and humanized anti-hapten antibody variable regions were combined with constant regions of human origin to form mono- or bispecific chimeric or humanized antibodies.
  • the generation of monospecific humanized anti-hapten antibodies and bispecific humanized anti-hapten antibodies that specifically bind a hapten as well as a different non-hapten target required (i) design and definition of amino acid and nucleotide sequences for such molecules, (ii) expression of these molecules in transfected cultured mammalian cells, and (iii) purification of these molecules from the supernatants of transfected cells.
  • a different non-hapten target e.g. receptor tyrosine kinases or IGF-1R
  • the humanized VH sequence was fused in frame to the N-terminus of CH1-hinge-CH2-CH3 of a human Fc-region of the subclass IgG1.
  • the humanized VL sequence was fused in frame to the N-terminus of human CLkappa constant region.
  • the anti-hapten antibody, a scFv or Fab fragment was fused in frame to the C-terminus of the heavy chain of previously described antibodies.
  • the applied anti-hapten scFv was further stabilized by introduction of a VH44-VL100 disulfide bond which has been previously described (e.g. Reiter, Y., et al., Nature biotechnology 14 (1996) 1239-1245).
  • Expression plasmids comprising expression cassettes for the expression of the heavy and light chains were separately assembled in mammalian cell expression vectors.
  • the transcription unit of the ⁇ -light chain is composed of the following elements:
  • the transcription unit of the yl-heavy chain is composed of the following elements:
  • DNA that contains coding sequences, mutations or further genetic elements was synthesized by Geneart AG, Regensburg.
  • DNA sequences were determined by double strand sequencing performed at SequiServe (SequiServe GmbH, Germany).
  • the Vector NTI Advance suite version 9.0 was used for sequence creation, mapping, analysis, annotation, and illustration.
  • the anti-hapten antibodies were expressed by transient transfection of human embryonic kidney 293 (HEK293) cells in suspension.
  • HEK293 human embryonic kidney 293
  • light and heavy chains of the corresponding mono- or bispecific antibodies were constructed in expression vectors carrying prokaryotic and eukaryotic selection markers as outlined above.
  • These plasmids were amplified in E. coli , purified, and subsequently applied for transient transfections. Standard cell culture techniques were used for handling of the cells as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc.
  • the cells were cultivated in appropriate expression medium at 37° C./8% CO 2 . On the day of transfection the cells were seeded in fresh medium at a density of 1-2 ⁇ 10 6 viable cells/mL.
  • the DNA-complexes with transfection reagents were prepared in Opti-MEM I medium (Invitrogen, USA) comprising 250 ⁇ g of heavy and light chain plasmid DNA in a 1:1 molar ratio for a 250 ml final transfection volume.
  • the monospecific or bispecific antibody containing cell culture supernatants were clarified 7 days after transfection by centrifugation at 14,000 g for 30 minutes and filtration through a sterile filter (0.22 ⁇ m). Supernatants were stored at ⁇ 20° C. until purification.
  • affinity HPLC chromatography was applied.
  • the cell culture supernatant containing mono- or bispecific antibody or derivatives thereof that bind to protein-A was applied to an Applied Biosystems Poros A/20 column in a solution comprising 200 mM KH 2 PO 4 , 100 mM sodium citrate, at pH 7.4.
  • Elution from the chromatography material was performed by applying a solution comprising 200 mM NaCl, 100 mM citric acid, at pH 2.5.
  • An UltiMate 3000 HPLC system (Dionex) was used.
  • the eluted protein was quantified by UV absorbance and integration of peak areas.
  • a purified IgG1 antibody served as a standard.
  • the recombinant antibody (or -derivatives) contained therein were purified from the supernatant in two steps by affinity chromatography using protein A-SEPHAROSETM affinity chromatography (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, the antibody containing clarified culture supernatants were applied on a MabSelectSuRe protein A (5-50 ml) column equilibrated with PBS buffer (10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were washed out with equilibration buffer.
  • PBS buffer 10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.4
  • the antibodies were eluted with 50 mM citrate buffer, pH 3.2.
  • the protein containing fractions were neutralized with 0.1 ml 2 M Tris buffer, pH 9.0.
  • the eluted protein fractions were pooled, concentrated with an Amicon Ultra centrifugal filter device (MWCO: 30 K, Millipore) and loaded on a Superdex200 HILOAD® 26/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM histidine, 140 mM NaCl, at pH 6.0.
  • the protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm with the OD at 320 nm as the background correction, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace et. al., Protein Science 4 (1995) 2411-2423. Monomeric antibody fractions were pooled, snap-frozen and stored at ⁇ 80° C. Part of the samples was provided for subsequent protein analytics and characterization.
  • the homogeneity of the antibodies was confirmed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.
  • a reducing agent 5 mM 1,4-dithiotreitol
  • Coomassie brilliant blue The NuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-20% Tris-Glycine gels).
  • polypeptide chains related to the IgG were identified after SDS-PAGE at apparent molecular sizes analogous to the calculated molecular weights. Expression levels of all constructs were analyzed by protein A. Average protein yields were between 6 mg and 35 mg of purified protein per liter of cell-culture supernatant in such non-optimized transient expression experiments.
  • the binding properties of the recombinant anti-biotin antibody derivatives were analyzed by biolayer interferometry (BLI) technology using an Octet QK instrument (Fortebio Inc.). This system is well established for the study of molecule interactions. BLi-technology is based on the measurement of the interference pattern of white light reflected from the surface of a biosensor tip and an internal reference. Binding of molecules to the biosensor tip is resulting in a shift of the interference pattern which can be measured. To analyze if the humanization procedure described above diminished the ability of the anti-biotin antibody to bind to biotin, the properties of the chimeric and the humanized versions of the antibody in their ability to bind to a biotinylated protein were compared directly.
  • Binding studies were performed by capturing anti-biotin antibody on anti-hulgG Fc antibody Capture (AHC) Biosensors (Fortebio Inc.). First, biosensors were incubated in an antibody solution with a concentration of 0.5 mg/ml in 20 mM histidine, 140 mM NaCl, pH 6.0 for 1 min. Thereafter, the biosensors were incubated for 1 min. in 1 ⁇ PBS pH 7.4 to reach a stable baseline. Binding was measured by incubating the antibody-coated biosensors in a solution containing biotinylated protein with a concentration of 0.06 mg/ml in 20 mM histidine, 140 mM NaCl, pH 6.0 for 5 min. Dissociation was monitored for 5 min. in 1 ⁇ PBS pH 7.4. The resulting binding curves for chimeric and humanized anti-biotin antibodies were compared directly.
  • AHC anti-hulgG Fc antibody Capture
  • the humanized version of the antibody showed equal or even better binding of the biotinylated antigen than the chimeric antibody. The same is true for the humanized antibody with the Cys mutation at Kabat position VH53.
  • the biotinylated protein showed residual unspecific binding to the biosensors which was reduced when the biosensors were coated with Herceptin, which does not bind biotin.
  • the functionality of the anti-biotin antibody was retained in its humanized variant (which is defined by the sequences as depicted in SEQ ID NO: 44 and 48, SEQ ID NO: 60 and 64).
  • Running and dilution buffer for the followed binding study was PBS-T (10 mM phosphate buffered saline including 0.05% Tween 20) pH 7.4.
  • the humanized anti-biotin antibody was captured by injecting a 2 nM solution for 60 sec at a flow rate of 5 ⁇ l/min.
  • Biotinylated siRNA was diluted with PBS-T at concentrations of 0.14-100 nM (1:3 dilution series). Binding was measured by injecting each concentration for 180 sec at a flow rate of 30 ⁇ l/min, dissociation time 600 sec. The surface was regenerated by 30 sec washing with a 3 M MgCl 2 solution at a flow rate of 5 ⁇ L/min.
  • the haptenylated compound was dissolved in H 2 O to a final concentration of 1 mg/ml.
  • Haptenylated payload and antibody were mixed to a 2:1 molar ratio (compound to antibody) by pipetting up and down and incubated for 15 minutes at RT.
  • the haptenylated compound was dissolved in 100% DMF to a final concentration of 10 mg/ml.
  • Haptenylated compound and antibody were mixed to a 2.5:1 molar ratio (compound to antibody) by pipetting up and down and incubated for 60 minutes at RT and 350 rpm.
  • Humanized and murine anti-digoxigenin antibody or bispecific anti-digoxigenin antibody derivatives were used as antibody components.
  • the Cy5-digoxigenin conjugate was dissolved in PBS to a final concentration of 0.5 mg/ml.
  • the antibody was used in a concentration of 1 mg/ml (about 5 ⁇ M) in a buffer composed of 20 mM histidine and 140 mM NaCl, pH 6.
  • Digoxigenylated Cy5 and antibody were mixed at a 2:1 molar ratio (digoxigenylated Cy5 to antibody). This procedure resulted in a homogenous preparation of complexes of defined composition.
  • the complexation reaction can be monitored by determining the fluorescence (650/667 nm) of the antibody-associated fluorophore on a size exclusion column.
  • the results of these experiments demonstrate that complexation only occurs if the antibody contains binding specificities for digoxigenin.
  • Antibodies without binding specificities for digoxigenin do not bind the digoxigenin-Cy5 conjugate.
  • An increasing signal can be observed for bivalent anti-digoxigenin antibodies until a digoxigenin-Cy5 conjugate to antibody ratio of 2:1. Thereafter, the composition dependent fluorescence signals reach a plateau.
  • Biotin-Cys-Cy5 For the generation of complexes of biotin-derivatized-Cy5 (Biotin-Cys-Cy5) containing a cysteinylated linker, 0.16 mg of Biotin-Cys-Cy5 were dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of the antibody was used in a concentration of 10.1 mg/ml (about 69 ⁇ M) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Cys-Cy5 and antibody were mixed at a 2.5:1 molar ratio (Biotin-Cys-Cy5 to antibody) and incubated for 60 min at RT, shaken at 350 rpm. The resulting conjugate was analyzed by SDS-PAGE as described in Example 6a. Detection of fluorescence was carried out as described in Example 6a.
  • Biotin-derivatized-Cy5 Biotin-Ser-Cy5
  • Biotin-Ser-Cy5 Biotin-Ser-Cy5
  • 0.61 mg of Biotin-Ser-Cy5 were dissolved in 20 mM histidine, 140 mM NaCl, pH 6.0 to a concentration of 10 mg/ml.
  • 18.5 mg of the humanized anti-biotin antibody was used in a concentration of 10 mg/ml (about 69 ⁇ M) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2.
  • Biotin-Ser-Cy5 and antibody were mixed at a 2.5:1 molar ratio (Biotin-Ser-Cy5 to antibody) and incubated for 60 min at RT, shaken at 350 rpm. The sample was then subjected to size exclusion chromatography using Superdex 200 16/60 high load prep grade column (GE Healthcare) with a flow rate of 1.5 ml/min and 20 mM histidine, 140 mM NaCl, pH 6.0 as the mobile phase. Peak fractions were collected and analyzed by SDS-PAGE for purity.
  • the resulting ratio of dye to antibody molecule was 2.17 which indicates that all antibody paratopes are saturated with Biotin-Cy5 molecules.
  • Digoxigenin-PYY(3-36) conjugate (11.57 mg, 4 ⁇ 10 ⁇ 6 mol, 2 eq.) was added in 4 portions of 2.85 mg within 1 h and incubated for another hour at room temperature.
  • the complexes were purified by size exclusion chromatography via a Superdex 200 26/60 GL column (320 ml) in 20 mM histidine, 140 mM NaCl, at pH 6.0 at a flow rate of 2.5 ml/min.
  • the eluted complex was collected in 4 ml fractions, pooled and sterilized over a 0.2 ⁇ m filter to give 234 mg of the complex at a concentration of 14.3 mg/ml.
  • the excess of polypeptide was removed by size exclusion chromatography via a Superose 6 10/300 GL column in 20 mM histidine, 140 mM NaCl, at pH 6.0 at a flow rate of 0.5 ml/min.
  • the eluted complex was collected in 0.5 ml fractions, pooled and sterilized over a 0.2 ⁇ m filter to give 4.7 mg of the complex at a concentration of 1.86 mg/ml.
  • the resulting haptenylated polypeptide-anti-hapten antibody complex was defined as monomeric IgG-like molecule via the occurrence of a single peak in a size exclusion chromatography.
  • the resulting complex was defined as monomeric IgG-like molecule, carrying two Digoxigenin-PYY derivatives per antibody molecule.
  • the defined composition of these peptide complexes was confirmed by size exclusion chromatography, which also indicated the absence of protein aggregates.
  • the defined composition (and 2:1 polypeptide to protein ratio) of these bispecific peptide complexes was further confirmed by SEC-MALS (Size exclusion chromatography-Multi Angle Light Scattering).
  • the sample was applied to a 4-12% Bis-Tris polyacrylamide-gel (NuPAGE®, Invitrogen) which was run for 35 min at 200V and 120 mA. Molecules that were separated in the polyacrylamide-gel were transferred to a PVDF membrane (0.2 ⁇ m pore size, Invitrogen) for 40 min at 25V and 160 mA. The membrane was blocked in 1% (w/v) skim milk in 1 ⁇ PBST (1 ⁇ PBS+0.1% Tween20) for 1 h at RT. The membrane was washed 3 ⁇ for 5 min in 1 ⁇ PBST and subsequently incubated with a streptavidin-POD-conjugate (2900 U/ml, Roche) which was used in a 1:2000 dilution. Detection of streptavidin-POD bound to biotin on the membrane was carried out using Lumi-Light Western Blotting Substrate (Roche).
  • the sample was applied to a 4-12% Bis-Tris polyacrylamide-gel (NuPAGE®, Invitrogen) which was run for 35 min at 200V and 120 mA. Molecules that were separated in the polyacrylamide-gel were transferred to a PVDF membrane (0.2 ⁇ m pore size, Invitrogen) for 40 min at 25V and 160 mA. The membrane was blocked in 1% (w/v) skim milk in 1 ⁇ PBST (1 ⁇ PBS+0.1% Tween20) for 1 h at RT. The membrane was washed 3 ⁇ for 5 min in 1 ⁇ PBST and subsequently incubated with a streptavidin-POD-conjugate (2900 U/ml, Roche) which was used in a 1:2000 dilution. Detection of streptavidin-POD bound to biotin on the membrane was carried out using Lumi-Light Western Blotting Substrate (Roche).
  • the generation of covalent conjugates of anti-hapten antibodies and haptenylated fluorescent dyes containing a cysteine-linker results in defined conjugates where a disulfide bridge is formed at a specific position between VH52bC in the CDR2 of the anti-hapten antibody and the cysteine in the linker between the hapten and the fluorescent dye.
  • the conjugation reaction was carried out in the presence of redox reagents. Dig-Cys-Ahx-Cy5 was dissolved in 20 mM histidine, 140 mM NaCl, pH 6.0. Solubilization was facilitated by drop wise addition of 10% (v/v) acetic acid. The final concentration was adjusted to 0.4 mg/ml.
  • the anti-digoxigenin antibody VH52bC in 20 mM histidine, 140 mM NaCl, pH 6.0 was brought to a concentration of 10 mg/ml.
  • An anti-digoxigenin antibody was used as a control and was treated the same way as anti-digoxigenin antibody VH52bC.
  • 4.7 nmol of each antibody was mixed with 2.5 molar equivalents of Dig-Cys-Ahx-Cy5. This was achieved by adding 11.7 nmol of this substance in 4 portions (2.9 nmol each) every 15 min. In between these additions, the samples were incubated at 25° C. while gently shaking.
  • each antibody—Dig-Cys-Ahx-Cy5 complex was transferred to buffer containing the following redox reagents: 3 mM DTE (Dithioerythritol)+10 mM GSSG (oxidized Glutathione), 0.3 mM DTE+1 mM GSSG and 0.03 mM DTE+0.1 mM GSSG. All samples were incubated for 15 min in these conditions. After the incubation, samples were split into half (0.34 nmol each) and prepared for SDS gel electrophoresis. For this, 4 ⁇ LDS sample buffer (Invitrogen) was added.
  • a reduced version was prepared by adding 10 ⁇ NuPAGE® sample reducing agent (Invitrogen). All samples were incubated at 70° C. for 5 min before electrophoresis on a 4-12% Bis-Tris polyacrylamide gel (NuPAGE®, Invitrogen) with 1 ⁇ MOPS buffer (Invitrogen). Cy5-related fluorescence in the gel was detected with a Lumilmager F1 device (Roche) at an excitation wavelength of 645 nm. After detection of fluorescence, the gel was stained with SimplyBlue SafeStain (Invitrogen). Gels are shown in FIG. 4 .
  • Dig-Cys-Cy5 was dissolved in 8.3 mM HCl, 10% (v/v) DMF to a final concentration of 3.25 mg/ml.
  • the anti-digoxigenin antibody VH52bC antibody in 20 mM histidine, 140 mM NaCl, pH 6.0 was brought to a concentration of 15 mg/ml.
  • anti-digoxigenin antibody was used as a control and was treated the same way as anti-digoxigenin antibody VH52bC.
  • conjugates of digoxigenin-derivatized-PYY-polypeptide containing a cysteinylated linker 1.4 mg of PEG3-PYY(PEG3-Cys-4Abu-Dig) were dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of the antibody was used in a concentration of 10 mg/ml (about 68 ⁇ M) in a buffer composed of 5 mM Tris-HCl, 1 mM EDTA, 1 mM GSH, 5 mM GSSG, pH 8.2.
  • PEG3-PYY(PEG3-Cys-4Abu-Dig) and antibody were mixed at a 2:1 molar ratio (PEG3-PYY(PEG3-Cys-4Abu-Dig) to antibody) and incubated for 60 min at RT, stirred at 100 rpm.
  • the resulting conjugate was analyzed by mass spectrometry. 43% of the detected species was identified as antibody coupled to 2 polypeptide molecules, 46% was antibody coupled to 1 polypeptide molecule and 11% was identified as uncoupled antibody.
  • hapten e.g. digoxigenin, fluorescein, biotin or theophylline
  • a suitable a reactive group such as e.g.
  • conjugates of anti-hapten antibodies with haptenylated compounds shall result in conjugates with defined stoichiometry and it shall be assured that the compound in these conjugates retains its activity.
  • the haptenylated compound was dissolved in 100% DMF to a final concentration of 10 mg/ml.
  • Haptenylated compound and anti-hapten antibody VH52bC/VH53C were mixed in a 2.5:1 molar ratio (compound to antibody) by pipetting up and down and incubated for 60 minutes at RT and 350 rpm.
  • a polypeptide conjugated to the hapten via a cysteine containing linker is termed hapten-Cys-polypeptide or polypeptide-Cys-hapten in the following.
  • the polypeptide may either have a free N-terminus or a capped N-terminus e.g. with an acetyl-group (Ac-polypeptide-Cys-hapten) or a PEG-residue (PEG-polypeptide-Cys-hapten).
  • a fluorescent dye conjugated to the hapten via a cysteine containing linker is termed dye-Cys-hapten or hapten-Cys-dye in the following.
  • Example 6a Samples were prepared exactly as described in Example 6a, with the difference that antibody-Dig-Cys-Ahx-Cy5 complexes were transferred to buffer containing either no redox compounds, 0.1 mM GSSG (oxidized glutathione) or 1 mM GSSG.
  • the resulting fluorescence-scanned and Coomassie stained polyacrylamide gels are shown in FIG. 5 . All three conditions show a similar specificity for site-specific disulfide bond formation ( FIG. 5 , top gels, lanes 1 A-C) with a low level of background reactions ( FIG. 5 , lanes 2 A-C). This confirms that formation of the disulfide bond can be accomplished without the need of reducing agents. This significantly stabilizes the antibody/reduces antibody disintegration, as only residual amounts of 3 ⁇ 4 antibodies ( ⁇ 1 ⁇ LC), HC-dimers ( ⁇ 2 ⁇ LC) and 1 ⁇ 2 antibodies (1 ⁇ HC+1 ⁇ LC) are detected in comparison to Example 6.
  • Biotin-Cys-Cy5 were dissolved in 100% DMF to a concentration of 10 mg/ml.
  • 1 mg of the humanized anti-biotin antibody VH53C was used in a concentration of 7.4 mg/ml (about 51 ⁇ M) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2.
  • Biotin-Cys-Cy5 and antibody were mixed at a 2.5:1 molar ratio (Ac-Biotin-Cys-Cy5 to antibody) and incubated for 60 min at RT, shaken at 350 rpm.
  • the resulting conjugate was analyzed by SDS-PAGE as described in Example 6a. Detection of fluorescence was carried out as described in Example 6a.
  • Theophyllin-Cys-Cy5 was generated as fluorescent payload, applying generally the synthesis and purification technologies that have been described for Digoxigenin-Cys-Cy5 or Biotin-Cys-Cy5, with the exception that the hapten has been exchanged against theophylline.
  • the composition of the Theophylline-Cys-Cy5 derivative that had been synthesized is shown in FIG. 16A ).
  • theophylline-binding antibodies were generated which contained a designed Cys at position 54 or 55 of the heavy chain variable region (anti-theophylline antibody-Cys). The purity of these antibodies is shown exemplarily for the Y54C variant in FIG. 16B ). These antibody derivatives were complexed with Theophylline-Cys-Cy5 and subsequently subjected to SDS-PAGE under non-reducing and reducing conditions. Under non-reducing conditions, disulfide-linked anti-theophylline-antibody complexed Cy5 was detected by its H-chain associated fluorescence within the gel. This is depicted in FIG.
  • polypeptides which are part of non-covalent hapten-polypeptide conjugates and in complexes with anti-hapten antibodies retain functionality (WO2011/003557, WO 2011/003780 and WO 2012/093068).
  • coupled peptides retain functionality also upon covalent disulfide-coupling, the biological activity of anti-digoxigenin antibody complexed polypeptides and their disulfide-conjugates with anti-digoxigenin antibody VH52bC were compared.
  • the therapeutically desired functionality of PYY-derived peptides is binding to and interfering with the signaling of its cognate receptor NPY2. Signaling via the NPY2 receptor is involved in and/or regulates metabolic processes.
  • Table 6 shows the results of cAMP-assays that were performed to assess the biological activity of PYY(3-36), its Y2 receptor specific modified analog moPYY, its antibody-complexed Dig-variant and its disulfide-conjugated Dig-Cys-derivative.
  • cAMP agonist assay the following materials were used: 384-well plate; Tropix cAMP-Screen Kit; cAMP ELISA System (Applied Biosystems, cat. #T1505; CS 20000); Forskolin (Calbiochem cat. #344270); cells: HEK293/hNPY2R; growth medium: Dulbecco's modified eagle medium (D-MEM, Gibco); 10% Fetal bovine serum (FBS, Gibco), heat-inactivated; 1% Penicillin/Streptomycin (Pen 10000 unit/mL: Strep 10000 mg/mL, Gibco); 500 ⁇ g/mL G418 (Geneticin, Gibco cat.
  • plating medium DMEM/F12 w/o phenol red (Gibco); 10% FBS (Gibco, cat. #10082-147), heat-inactivated; 1% Penicillin/Streptomycin (Gibco, cat. #15140-122); 500 ⁇ g/mL G418 (Geneticin, Gibco, cat. #11811-031).
  • HEK293/hNPY2R 10,000 cells/well
  • Multi-drop dispenser 50 microliters of cells (HEK293/hNPY2R—10,000 cells/well) were transferred into the 384-well plate using Multi-drop dispenser. The plates were incubated at 37° C. overnight. On the second day, the cells were checked for 75-85% confluence. The media and reagents were allowed to come to room temperature. Before the dilutions were prepared, the stock solution of stimulating compound in dimethyl sulphoxide (DMSO, Sigma, cat#D2650) was allowed to warm up to 32° C. for 5-10 min. The dilutions were prepared in DMEM/F12 with 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX, Calbiochem, cat#410957) and 0.5 mg/mL BSA.
  • DMSO dimethyl sulphoxide
  • the final DMSO concentration in the stimulation medium was 1.1% with Forskolin concentration of 5 ⁇ M.
  • the cell medium was tapped off with a gentle inversion of the cell plate on a paper towel. 50 ⁇ L of stimulation medium was placed per well (each concentration done in four replicates). The plates were incubated at room temperature for 30 min, and the cells were checked under a microscope for toxicity. After 30 min of treatment, the stimulation media was discarded and 50 ⁇ L/well of Assay Lysis Buffer (provided in the Tropix kit) was added. The plates were incubated for 45 min @ 37° C. 20 ⁇ L of the lysate was transferred from stimulation plates into the pre-coated antibody plates (384-well) from the Tropix kit.
  • the objective of the described peptide modification technology is to improve the therapeutic applicability of peptides.
  • Major bottlenecks for therapeutic application of peptides are currently limited stability in vivo and/or short serum half-life and fast clearance.
  • the PK parameters of antibody conjugates of fluorophores were determined in vivo and compare with the PK of non-covalent antibody-fluorophore complexes.
  • the anti-biotin antibody VH53C was covalently conjugated to the biotinylated fluorophore Biot-Cys-Cy5, (ii) a non-covalent complex of the anti-biotin antibody with biotinylated fluorophore Biot-Cy5 was generated, (iii) the covalently conjugated and the non-covalently complexed compounds were applied to animals and (iv) the serum concentrations of the compounds over time in these animals was measured by determination of the fluorescence of Cy5 (A650), and that of the corresponding antibody by an ELISA method that specifically detects the humanized antibody.
  • Cy5 related fluorescence in serum samples were measured in 120 ⁇ l quartz cuvettes at room temperature using a Cary Eclipse Fluorescence Spectrophotometer (Varian). Excitation wavelength was 640 nm,
  • Emission was measured at 667 nm. Serum samples were diluted in 1 ⁇ PBS to reach an appropriate range of Emission intensity. Blood serum of an untreated mouse in the same dilution in 1 ⁇ PBS as the respective sample was used as a blank probe and did not show any fluorescence signal.
  • FIGS. 7A-7B show the results of an analysis employing covalent conjugates, non-covalent complexes and non-complexed hapten-Cy5.
  • the data is shown as relative (%) levels of Cy5-mediated fluorescence normalized to the (peak) serum levels 5 min after injection.
  • non-complexed Biotin-Ser-Cy5 disappears rapidly from the serum.
  • One hour after injection only 6% of the fluorescence that was applied and detectable after 5 minutes in the serum was still detectable.
  • 2 hrs., 4 hrs. and 24 hrs. after injection Cy5-mediated signals were not detectable.
  • human IgG1 antibodies in serum samples were captured on a solid phase (Maxisorb® microtiter plate, NUNC-ImmunoTM) coated with an anti-human kappa-chain monoclonal IgG antibody.
  • Serum samples were diluted 1:10 5 and 1:10 6 and 100 ⁇ l of these dilutions were added to the wells. After incubation, wells were washed 3-times with 300 ⁇ l PBS/0.05% Tween 20 each.
  • Detection of human IgG antibodies was carried out by first adding 100 ⁇ l of anti-human C H 1-domain IgG which is digoxigenylated at the C-terminus at a concentration of 0.25 ⁇ g/ml. After washing 3-times with 300 ⁇ l of 1 ⁇ PBS/0.05% Tween 20 each, 100 ⁇ l of anti-digoxigenin antibody Fab-fragment conjugated to horse-radish peroxidase (HRP) was added at a concentration of 25 mU/ml. Finally, per well 100 ⁇ l of ABTS® were added. After 30 min. incubation at ambient temperature, the extinction (OD) was measured at 405 nm and 492 nm [405/492] in a commercial microtiter plate ELISA Reader (e.g. Tecan Sunrise).
  • HRP horse-radish peroxidase
  • FIGS. 7A-7B show the Bio-Cy5 serum levels as well as the serum levels of human IgG in mice treated with antibody-biotin-Cy5-complexes and -conjugates.
  • the data is shown as relative (%) human IgG levels normalized to the (peak) serum levels 5 min. after injection.
  • the relative human IgG serum levels of both antibody-hapten-complexes and -conjugates are in-line with the relative fluorescence measured for the antibody-hapten conjugates.
  • the Biotin-Cys-Cy5 compound shows a similar in vivo stability as the antibody it is conjugated to, which means that antibody-hapten conjugates stay intact in vivo. This is clearly not the case for antibody-hapten complexes for which the relative Cy5-mediated fluorescence decreases faster than the relative human IgG serum levels. This means that the complexes release the payload over time in vivo.
  • haptenylated compounds are significantly increased when bound by an anti-hapten antibody.
  • antibody-hapten complexes are not completely stable in vivo as the decrease of the hapten-Cy5 serum levels is faster than the decrease of antibody serum levels. This is not the case for antibody-hapten-Cy5 conjugates, which show a similar in vivo behavior as normal IgG antibodies.
  • mice To analyze the influence on PK parameters of antibody-complexation and antibody conjugation of the digoxigenylated polypeptide, 32.1 nmol of the polypeptide, or of the corresponding antibody non-covalently complexed polypeptide in 20 mM histidine/140 mM NaCl pH 6.0 were applied to 2 female mice (strain NRMI) for each substance. The mice had a weight of 23 g and 25 g for MAK-DIG-PYY and 28 g and 26 g for DIG-PYY. About 0.1 ml blood samples were collected after the following time points: 0.08 h, 2 h and 24 h for Mouse 1 and 0.08 h, 4 h 24 h for Mouse 2. Serum samples of at least 40 ⁇ l were obtained after 1 h at RT by centrifugation (9300 ⁇ g, 3 min, 4° C.). Serum samples were stored at ⁇ 80° C.
  • each serum sample was diluted in 18 ⁇ l 20 mM histidine/140 mM NaCl pH 6.0, mixed with 6.7 ⁇ l of 4 ⁇ LDS sample buffer and 3 ⁇ l of 10 ⁇ sample reducing agent (NuPAGE®, Invitrogen) for 5 min at 95° C.
  • sample reducing agent NuPAGE®, Invitrogen
  • 2 ⁇ l of serum of an untreated mouse of the same strain was used. Samples were applied to a 4-12% Bis-Tris Gel (NuPAGE®, Invitrogen) which was run at 200 V/120 mA for 20 minutes using 1 ⁇ MES (Invitrogen) as a running buffer.
  • Digoxigenylated polypeptides were subsequently detected on the membrane with an anti-digoxigenin antibody.
  • anti-digoxigenin antibody was applied to the membranes in a concentration of 13 ⁇ g/ml in 10 ml of 1% skim milk/PBST for 2 h at RT. Membranes were washed for 3 ⁇ 5 min in 1 ⁇ PBST.
  • Anti-mouse IgG Fab-fragments coupled to POD from the LumiLight PLUS Western Blotting Kit (Roche) was applied in a 1:25 dilution in 10 ml of 1% skim milk/PBST for 1 h at RT. Membranes were washed 3 ⁇ 5 min with 1 ⁇ PBST. Detection was carried out by incubating the membranes in 4 ml LumiLight Western Blotting substrate for 5 min at RT. Chemiluminescence was detected with the Lumilmager F1 (Roche) with an exposure time of 20 min.
  • FIG. 8 The results of these analyses are shown in FIG. 8 .
  • the presence/amount of the digoxigenin polypeptide in murine serum at different time points has been determined. Mice that had received antibody complexed peptides ( FIG. 8 left) showed strong signals at the earliest time point (5 min after administration). These signals were clearly assignable as shown by the size and location on the blot of the controls.
  • polypeptide-associated signals were strongest at the early time points and decreased over time. Nevertheless, polypeptide was still detectable with good signals at all time points and even 24 hrs. after administration.
  • FIG. 8 shows in the right that under normal exposure conditions, no free polypeptide is visible on the blot. Contrast enhancement of the blot revealed the presence of some polypeptide 5 min after administration, however only in trace amounts. At later time points, no defined polypeptide band was detectable.
  • non-complexed polypeptide has a very short half-life in the serum of mice. Mice that received the same polypeptides but in antibody complexed form, show presence of these polypeptides in the serum for an increased period of time. Twenty four hrs. after injection polypeptide can be determined in the serum of these mice.
  • the PK parameters of anti-digoxigenin antibody-Digoxigenin-Cy5 complexes, as well as of the covalently linked [anti-digoxigenin antibody-Cys]-[Digoxigenin-Cys-Cy5] conjugates were determined in vivo. Therefore, Digoxigenin-Cy5 was determined using its fluorescence (A650), and the corresponding antibody was determined by an ELISA method that specifically detects the humanized antibody. Digoxigenin-Cy5 was applied as low molecular weight ‘surrogate’ for hapten-coupled peptides because its fluorescent properties allow easy and accurate detection in the serum.
  • Digoxigenin-Cy5 or antibody-complexed or additionally antibody-Cys-linked Digoxigenin-Cy5 were injected intravenously into female NRMI mice, followed by collection of blood at 0.08 h, 2 h, 4 h and 24 h.
  • the PK of a complex of anti-biotin antibody with Biotin-Cy5, as well as that of the covalently linked conjugate [anti-biotin-antibody-Cys]-[Biotin-Cys-Cy5] in vivo were determined.
  • the presence of Cy5 was determined non-invasive by optical imaging of the eye of animals, which revealed the fluorescence of Cy5 in the capillaries.
  • the Cy5-mediated fluorescence values that we detected in the eye of mice 10 min. after injection was set as 100% value, and fluorescence values measured at subsequent time points were expressed relative thereto. The results of these experiments are shown in FIG.
  • non-complexed Biotin-Cy5 by itself has a low serum half-life and low exposure levels. Antibody-complexed compound which was not covalently linked was detectable at much higher levels and with an extended half-life. Furthermore, covalently linked payloads displayed a greater PK prolongation, and higher serum levels compared to the non-covalently linked complexes. This indicates that hapten-complexed disulfide-linked payloads are more stable in the circulation, and can reach higher exposure levels, than non-covalent complexed payloads.
  • the concept can be expanded to further haptens or other entities that capture compounds and connect them to the targeting module.
  • mono- or bispecific antibodies that bind digoxigenin or other haptens can be applied to stabilize and PK-optimize therapeutic polypeptides.
  • Prerequisites for application as polypeptide capturing modules are (i) that coupling of compounds to the hapten does not severely interfere with polypeptide activity and (ii) the possibility of effective binding/complexation of the antibody to haptenylated compounds.
  • Hapten-directed binding is a prerequisite for the efficient covalent coupling of haptenylated dyes or polypeptides with an anti-hapten cysteinylated antibody.
  • Biotin-Cys-Cy5 was incubated with humanized anti-digoxigenin antibody and humanized anti-digoxigenin antibody VH53C. Incubation of Biotin-Cys-Cy5 with humanized anti-biotin antibody and humanized anti-biotin antibody VH53C was carried out as a control reaction.
  • Biotin-Cys-Cy5 were dissolved in 100% DMF to a concentration of 10 mg/ml. 0.7 mg of each antibody was used in a concentration of 6.7 mg/ml (about 46 ⁇ M) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Cys-Cy5 and antibodies were mixed at a 2.5:1 molar ratio (Ac-Biotin-Cys-Cy5 to antibody) and incubated for 60 min at RT, shaken at 350 rpm. The resulting complex/conjugate was further analyzed by SDS-PAGE and subsequent detection of Cy5-related fluorescence in the polyacrylamide-gel.
  • the non-reduced samples show covalent site-specific disulfide bond formation for humanized anti-biotin antibody VH53C ( FIG. 9 , lane 4) with very low background fluorescence signal when humanized anti-biotin antibody without a cysteine in CDR2 was used ( FIG. 9 , lane 3).
  • Biotin-Cys-Cy5 was also covalently coupled to humanized anti-digoxigenin antibody VH52bC ( FIG. 9 , lane 2) with a low background signal when humanized anti-digoxigenin antibody was used ( FIG. 9 , lane 1), but with significantly lower efficiency. This can be deduced from the excess Biotin-Cys-Cy5 that is detected on the bottom of the gel (arrows).
  • Hapten-Directed Binding is a Prerequisite for the Efficient Covalent Coupling of Haptenylated Dyes or Polypeptides with an Anti-Hapten Cysteinylated Antibody
  • the non-haptenylated peptide Ac-PYY(PEG3-Cys-4Abu-NH2) (Biosynthan 1763.1, SEQ ID NO: 178) was incubated with humanized anti-digoxigenin antibody VH52bC and humanized anti-digoxigenin antibody. 1.4 mg of Ac-PYY(PEG3-Cys-4Abu-NH2) were dissolved in 100% DMF to a concentration of 10 mg/ml.
  • each antibody was used in a concentration of 11-13 mg/ml (about 75-89 ⁇ M) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2.
  • Ac-PYY(PEG3-Cys-4Abu-NH2) and antibodies were mixed at a 2.1:1 molar ratio (Ac-PYY(PEG3-Cys-4Abu-NH2 to antibody)).
  • the peptide was added in 3 portions while the solution was stirred at 500 rpm with a stirrer bar. Between each addition, samples were incubated for 5 min at 200 rpm. After addition of the last portion, samples were incubated for 1 h at RT and 200 rpm.
  • the resulting complex/conjugate was defined as 97% monomeric IgG-like molecule and 3% dimeric soluble aggregates for the Ac-PYY(PEG3-Cys-4Abu-NH2): humanized anti-digoxigenin antibody VH52bC conjugate and as 100% monomeric for the Ac-PYY(PEG3-Cys-4Abu-NH2): humanized anti-digoxigenin antibody complex via size exclusion chromatography. Furthermore, the resulting complex/conjugate was analyzed by mass spectrometry.
  • Hapten-binding modules for covalent compound/payload coupling may be composed of ‘standard’ antibodies such as IgGs which contain extra cysteines that enable covalent attachment of haptenylated compounds/payloads.
  • the method as reported herein introduces the required functionalities (cysteines) within folded domains, whose structure and sequence provide the basis for antibody functionality. Correct formation of defined disulfide bonds within as well as between the domains of antibodies is essential for the formation and maintenance of the correct structure and functionality.
  • FIG. 10A shows the disulfide pattern that is required to form functional binding arms such as Fabs of unmodified antibodies
  • FIG. 10B shows the disulfide pattern which is necessary to maintain structure and functionality of the VH52cB/VH53C mutated antibody derivative.
  • FIG. 10C and FIG. 10D show that the additions of the extra cysteines generate possibilities to form incorrect disulfides within the VH domains during the biosynthesis of such molecules.
  • Another potential problem is that VH and VL domains become assembled within the secretory pathway to one Fv fragment.
  • the secretory pathway involves redox-shuffling conditions and disulfide forming and—shuffling enzymes. Therefore, the potential to introduce incorrect disulfides by addition of the VH52bC/VH53C mutation may ‘spread’ also to disulfides of the light chain (exemplarily shown in FIG. 10E ). This does further enhance the risk to obtain/generate improperly folded non-functional molecules. It is therefore quite surprising that—despite of these risks—good amounts of homogeneous functional antibody derivatives that contain the VH52bC/VH53C mutation could be expressed and obtained, and which are capable to covalently connect to haptenylated compounds/payloads.
  • Hapten-binding modules for covalent compound/payload coupling can consist of ‘standard’ antibodies such as IgGs. Alternatively, they may be modified entities such as recombinant Fv or Fab fragments, or derivatives thereof. Single-chain Fv fragments are frequently applied as alternative to full lengths antibodies, especially in applications where small module size is required, or where additional binding modules are desired to generate bi- or multispecific antibody derivatives.
  • One example for anti-hapten Fv-derived entities that have been generated is a disulfide-stabilized single-chain Fv which bind to and covalently connects digoxigenylated compounds/payloads.
  • the disulfide-stabilized single-chain Fv with Dig-binding specificity was generated by connecting anti-digoxigenin antibody VH and VL domains via a flexible Gly and Ser rich linker to each other. These VH and VL domains harbored in addition cysteine mutations in positions 44 of VH and position 100 of VL (positions according to Kabat et al.). These additional cysteines form a stable intermolecular disulfide bond between VH and VL. This stabilizes the scFv, as previously described (e.g. Reiter, Y., et al., Nature Biotechnology 14 (1996) 1239-1245).
  • Disulfide connections between non-matching cysteines will generate misfolded instable and non-functional entities.
  • a disulfide-stabilized Fv fragment contains 6 cysteines
  • 21 different disulfide connections can theoretically be formed—but only the right combination of 3 defined disulfides will form a functional stabilized dsscFv.
  • the stabilized dsscFv that is desired contains two defined intradomain disulfides (one each in VH and VL), one defined interdomain disulfide (between VH and VL), and furthermore one free cysteine for haptenylated compound/payload coupling (in VH at position 52b/53).
  • VH44C is necessary for forming the correct inter-domain disulfide and this disulfide most likely without being bound by this theory forms after independent folding and assembly of VH and VL. Proximity of VH44C and VH52bC/VH53C aggravates the risk that the intradomain disulfide does not form in a correct manner. But it has been found that functional disulfide stabilized single-chain Fv modules that bind digoxigenin and that are simultaneously capable to covalently connect to digoxigenylated payloads can be produced. The composition of the disulfide-stabilized single-chain Fv molecule that contains the correct disulfides and the free cysteine in the correct position and its comparison to the undesired incorrectly folded molecules is shown in FIG.
  • Bispecific antibodies were generated that contain hapten-binding antibody modules for covalent compound/payload coupling. These antibodies additionally contain binding modules that enable targeting to other antigens. Applications for such bispecific antibodies include specific targeting of haptenylated compounds/payloads to cells or tissues that carry the targeting antigen.
  • One example for such molecules that was generated is a bispecific antibody with binding regions that recognize the tumor associated carbohydrate antigen LeY, and simultaneously with disulfide-stabilized Fvs which bind and covalently connect digoxigenylated compounds/payloads.
  • disulfide-stabilized single-chain Fvs were connected via flexible Gly and Ser rich connector peptides to the C-termini of the CH3 domains of a LeY antibody, resulting in tetravalent molecules with two LeY binding arms and additionally two digoxigenin binding entities.
  • the digoxigenin-binding entities harbored a VH44-VL100 disulfide bond which has been previously described (e.g. Reiter, Y., et al., Nature Biotechnology 14 (1996) 1239-1245).
  • the digoxigenin binding entity contained in addition the VH52bC mutation for covalent coupling.
  • the sequences that encode the light chain and the modified heavy chain of this LeY-Dig antibody derivative are listed under SEQ ID NO: 191 and SEQ ID NO: 192.
  • the composition of the LeY-Dig bispecific antibody derivative as delivery vehicle for covalently coupled compounds/payloads is shown schematically in FIG. 12 .
  • FIG. 13A shows the presence of modified H-chain and L-chain of this LeY-Dig (52bC) bispecific antibody in cell culture supernatants, visualized in Western Blot analyses that detect antibody L-chains and H chains.
  • FIG. 13B demonstrates the homogeneity of these antibodies after purification by SDS-PAGE in the presence of a reducing agent. Staining of the SDS-PAGE with Coomassie brilliant blue visualizes polypeptide chains related to the IgG with the apparent molecular sizes analogous to their calculated molecular weights.
  • 13C shows the SEC profile of the LeY-Dig(52bC) bispecific antibody after Protein A purification, demonstrating homogeneity and lack of aggregates in the protein preparations.
  • bispecific antibodies which contain targeting modules as well as modules for covalent coupling of haptenylated compounds/payloads can be generated and purified to homogeneity.
  • the protein structure of murine anti-Biotin antibody Fab-fragment was determined in complex with biocytinamid. Therefore, crystals of the Fab-fragment were grown in 0.8 M Succinic Acid, followed by charging of the antibody crystals with Biocytinamid (diluted to 10 mM working concentration in crystallization solution, applied to the crystals in the crystallization droplet). Crystals were washed three times with 2 ⁇ l of 10 mM Biocytinamid solution and were finally incubated for 16 hrs. with Biocytinamid at 21° C., harvested with 15% Glycerol as cryoprotectant and flash frozen in liquid nitrogen. Processed diffraction images yielded a protein structure at 2.5 ⁇ resolution.
  • Biotin binds into a surface pocket which is flanked by charged regions that composed of amino acids from the CDR regions.
  • the complexed hapten is positioned in close proximity to a negatively charged cluster of amino acids.
  • Biotin which—as hapten—is derivatized for payload coupling at its carboxyl group binds with good efficacy as there is no charge repulsion at this position (due to the lack of the COOH group).
  • free (normal) biotin cannot bind efficient to the antibody because its carboxyl group would be in close proximity to this negative charge cluster, and hence becomes repulsed.
  • the bispecific antibodies recognize tumor associated cell surface antigens such as LeY and simultaneously bind haptens such as e.g. digoxigenin (Dig).
  • FIG. 20A shows the composition of the bispecific antibodies, based upon a previously described full length.
  • the cell surface antigen binding functionalities are located in the two Fab arms of the IgG moiety, two additional scFvs recombinantly fused to the C-termini of the heavy chains have hapten binding activity.
  • the scFv modules carry additional stabilizing interchain disulfide bonds to stabilize the Fv and reduce aggregation (VHCys44 to VLCys100).
  • the anti-hapten antibody has an artificial cysteine residue at position VH52b or VH53 according to the Kabat numbering depending on the actual length of the CDR2 of the respective antibody, to enable the formation of a covalent disulfide bond between the bispecific anti-hapten antibody and the polypeptide toxin.
  • the bispecific antibodies were transiently produced in HEK293 cells in suspension and purified from culture supernatants as previously described (see Metz et al supra, WO 2012/093068). Plasmids encoding light and heavy chains or of the Fab-Fv fusions were co-transfected into HEK293 suspension cells which were cultivated in serum free medium. Supernatants were clarified seven days after transfection by centrifugation and 0.22 ⁇ m filtration. The bispecific antibodies were purified by protein followed by SEC (Superdex200 HiLoad 26/60, GE Healthcare) equilibrated with 20 mM histidine, 140 mM NaCl, at pH 6.0. Protein concentrations were determined by optical density at 280 nm with 320 nm as background, homogeneity of purified proteins was confirmed by SDS-PAGE.
  • composition, purity and homogeneity of the resulting bispecific antibody preparations (SEC and SDS PAGE) that were applied in this study are shown in FIG. 20B .
  • Hapten-binding modules for covalent coupling with a polypeptide toxin are composed of ‘standard’ antibodies such as IgGs which contain one or more extra cysteine residues for the formation of a disulfide bond with the haptenylated polypeptide toxin.
  • the method as reported herein introduces the required functionalities (cysteines) within folded domains, whose structure and sequence provide the basis for antibody functionality. Correct formation of defined disulfide bonds within as well as between the domains of antibodies is essential for the formation and maintenance of the correct structure and functionality.
  • FIG. 10A shows the disulfide pattern that is required to form functional binding arms such as Fabs of unmodified antibodies, and FIG.
  • FIGS. 10C and 10D show that the additions of the artificial cysteine residues generate possibilities to form incorrect disulfides within the VH domains during the biosynthesis of such molecules.
  • VH and VL domains become assembled within the secretory pathway to one Fv fragment.
  • the secretory pathway involves redox-shuffling conditions and disulfide forming and -shuffling enzymes. Therefore, the potential to introduce incorrect disulfides by addition of the VH52bC/VH53C mutation may ‘spread’ also to disulfides of the light chain (exemplarily shown in FIG. 10E ). This does further enhance the risk to obtain/generate improperly folded non-functional molecules.
  • hapten-binding modules for covalent polypeptide toxin coupling can consist of ‘standard’ antibodies such as IgGs. Alternatively, they may be antibody fragments such as Fv or Fab fragments, or derivatives thereof. Single-chain Fv fragments are frequently applied as alternative to full lengths antibodies, especially in applications where small module size is required, or where additional binding modules are desired to generate bi- or multispecific antibody derivatives.
  • anti-hapten Fv-derived entities that have been generated is a disulfide-stabilized single-chain Fv which bind to and covalently connects digoxigenylated polypeptide toxins.
  • the disulfide-stabilized single-chain Fv with digoxigenin-binding specificity was generated by connecting anti-digoxigenin antibody VH and VL domains via a flexible GS-linker to each other. These VH and VL domains harbored in addition cysteine mutations in positions 44 of VH and position 100 of VL (positions according to Kabat et al.). These additional cysteines form a stable intermolecular disulfide bond between VH and VL. This stabilizes the scFv, as previously described (e.g. Reiter, Y., et al., Nature Biotechnology 14 (1996) 1239-1245).
  • one single defined correct interdomain disulfide needs to be formed.
  • Disulfide connections between non-matching cysteines will generate misfolded instable and non-functional entities.
  • a disulfide-stabilized Fv fragment contains 6 cysteines
  • 21 different disulfide connections can theoretically be formed—but only the right combination of three defined disulfides will form a functional stabilized single-chain disulfide stabilized Fv-fragment (dsscFv). This challenge is aggravated upon addition of another free cysteine into the VH domain.
  • the disulfide stabilized Fv fragment (dsscFv) that is desired contains two defined intradomain disulfides (one each in VH and VL), one defined interdomain disulfide (between VH and VL), and furthermore one free cysteine for haptenylated compound/payload coupling (in VH at position 52b/53).
  • dsscFv disulfide stabilized Fv fragment
  • two defined intradomain disulfides one each in VH and VL
  • one defined interdomain disulfide between VH and VL
  • furthermore one free cysteine for haptenylated compound/payload coupling in VH at position 52b/53.
  • VH52b/VH53 is located in close proximity to the VH44 cysteine which is also a not naturally occurring cysteine but a mutation introduced for disulfide stabilization.
  • VH44C is necessary for forming the correct inter-domain disulfide. Without being bound by this theory this disulfide bond most likely forms after independent folding and assembly of VH and VL. Proximity of VH44C and VH52bC/VH53C aggravates the risk that the intradomain disulfide does not form in a correct manner.
  • Pseudomonas exotoxin is a 66 kDa protein which binds with its N-terminal domain I to eukaryotic cells, internalizes, becomes proteolytically processed in domain II and releases the C-terminal domain III into the cytoplasm. This domain ADP-ribosylates eEF2, which causes inhibition of protein synthesis and subsequent cell death). Truncated variants including those used herein as polypeptide toxin are shown in FIG. 21 .
  • the molecule NLysPE38 has the cell binding domain I and domain IB deleted (Weldon, J. E. and Pastan, I., FEBS Journal 278 (2011) 4683-4700; Debinski, W. and Pastan, I., Cancer Res.
  • NLysPE38 contains a lysine close to its N-terminus (N-Lys) which can be chemically modified by NHS-chemistry. It was recently shown that—within the context of dsscFv-fusions—most of domain II can also be deleted without loss of potency as long as the processing site is retained (Weldon, J. E. and Pastan, I., FEBS Journal 278 (2011) 4683-4700; Hansen, J. K., et al., J. Immunother. 33 (2010) 297-304; Pastan, I., et al., Leukemia & Lymphoma 52 (2011) Supp.
  • Truncated toxins still contain lysine residues in domain III.
  • the previously described toxin NlysPE38QQR has the lysines of domain III replaced by glutamine or arginine (Debinski, W. and Pastan, I., Cancer Res. 52 (1992) 5379-5385; Debinski, W. and Pastan, I., Bioconjug. Chem. 5 (1994) 40-46), to reduce the risk of inactivation of domain III by amine-modifying reagents such as NHS.
  • NLysPE25SQ ⁇ is a rather small toxin moiety that contains only one lysine at its N-terminus.
  • the primary amine of this lysine (and that of the N-terminus) can be modified by NHS-reagents without affecting other positions of the toxin.
  • NLysPE25SQ ⁇ (PE25) was produced in E. coli and purified from the periplasm by anion-exchange and size exclusion chromatography to remove aggregates and smaller sized impurities as previously described for NLysPE38 (Debinski, W. and Pastan, I., Cancer Res. 52 (1992) 5379-5385; Debinski, W. and Pastan, I., Bioconjug. Chem.
  • NLysPE25SQ ⁇ the active ester digoxigenin-NHS was applied for modification of primary amino groups of lysines.
  • the N-terminus of the proteins may also serve as target for NHS-mediated digoxigenin-conjugation.
  • a short but flexible linker was placed between digoxigenin and NHS, i.e. between digoxigenin and the polypeptide toxin.
  • NLysPE25SQ ⁇ ⁇ is termed PE25.
  • a cysteine was engineered into PE25 either before or after the lysine residue that is used for hapten-conjugation.
  • These PE derivatives were produced and purified in the same manner as PE25.
  • the PE25 variant that has the artificial polypeptide cysteine residue before the lysine residue that is used for hapten-conjugation is termed NCK-PE25 and the PE25 variant that has the artificial polypeptide cysteine residue after the lysine residue that is used for hapten-conjugation is termed NKC-PE25.
  • a comparison of the sequences of PE25, NCK-PE25 and NKC-PE25 is shown in FIG. 21C .
  • the sequence of PE25 is listed in SEQ ID NO: 194.
  • the sequence of the S-PE25 derivative which has the lysine replaced by a serine is listed in SEQ ID NO: 195.
  • the sequence of NKC-PE25 is listed in SEQ ID NO: 196.
  • the sequence of NCK-PE25 is listed in SEQ ID NO: 197.
  • PE25 derivatives were reacted with digoxigenin-carboxymethyl-NHS ester (DE3836656, Metz et al. supra).
  • Small non-reacted compounds and reaction products (NHS) were subsequently separated from the digoxigenylated 25 kDa polypeptide toxin by passing the reaction through a PD10 column, followed by sterile filtration.
  • digoxigenylated polypeptide toxins Prior to antibody coupling, digoxigenylated polypeptide toxins were reduced with DTE followed by subsequent removal of the reducing agent by passing again through a PD10 column. Subsequently, complexes containing bispecific antibody and digoxigenylated polypeptide toxin were generated by incubating digoxigenylated polypeptide toxins for 10 min.
  • FIGS. 22B and 22C A model with the relative sizes and composition of the components of digoxigenylated-PE25, NCK-PE25 and NKC-PE25 complexed and stably disulfide bonded with the digoxigenin-binding antibody moiety is shown in FIGS. 22B and 22C .
  • MCF-7 cells were exposed to covalent conjugates of bispecific antibody and polypeptide toxin.
  • MCF7 expresses LeY antigen on their surface and are sensitive to PE-derived immunotoxins that bind the LeY (see Metz, S., et al. supra). Viability and proliferation of these cells was assessed by assays that determine metabolic activity or cell proliferation, both are method well known to experts in the field. To determine the viability of cells upon expose to toxins, cellular ATP content (reflects metabolic activity) was determined with Cell Titer Glo (CTG) assays 48 hrs. or 72 hrs.
  • CCG Cell Titer Glo
  • Covalent complexes as reported herein can be delivered to and into cells by bispecific cell targeting antibodies (bsAbs) that comprise an anti-hapten binding specificity.
  • bsAbs bispecific cell targeting antibodies
  • the polypeptides that are delivered by the cell targeting bsAb may need to be released within the target cells.
  • LeY-Dig(52bC) bispecific antibody that bind to the LeY antigen and carries anti-Biotin binding entities as disulfide-stabilized single-chain Fv additions was used. Incubation with Biotin-Cys-Cy5 generated covalent disulfide-conjugated complexes.
  • the LeY antigen is abundant on breast cancer cells, internalizes, and LeY-binding antibody derivatives have previously been shown to deliver payloads to and into cells (such as MCF7). Confocal microscopy analyses using Alexa-labeled secondary antibodies to detect bsAb and fluorescence to detect Cy5 showed that the covalent conjugate bound to and internalizes into MCF7 ( FIG.

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